WO2018077926A1 - Anticorps monoclonaux se liant à l'isoforme transmembranaire cd160 - Google Patents

Anticorps monoclonaux se liant à l'isoforme transmembranaire cd160 Download PDF

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WO2018077926A1
WO2018077926A1 PCT/EP2017/077261 EP2017077261W WO2018077926A1 WO 2018077926 A1 WO2018077926 A1 WO 2018077926A1 EP 2017077261 W EP2017077261 W EP 2017077261W WO 2018077926 A1 WO2018077926 A1 WO 2018077926A1
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antibody
seq
monoclonal antibody
cells
amino acid
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PCT/EP2017/077261
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English (en)
Inventor
Armand Bensussan
Bruno Robert
Pierre Martineau
Myriam CHENTOUF
Anne Marie-Cardine
Jérôme GUSTINIANI
Original Assignee
INSERM (Institut National de la Santé et de la Recherche Médicale)
Université Paris Diderot - Paris 7
Université De Montpellier
Institut Jean Godinot
Institut Régional Du Cancer De Montpellier
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Application filed by INSERM (Institut National de la Santé et de la Recherche Médicale), Université Paris Diderot - Paris 7, Université De Montpellier, Institut Jean Godinot, Institut Régional Du Cancer De Montpellier filed Critical INSERM (Institut National de la Santé et de la Recherche Médicale)
Priority to AU2017351764A priority Critical patent/AU2017351764A1/en
Priority to US16/343,889 priority patent/US11186635B2/en
Priority to CA3080270A priority patent/CA3080270A1/fr
Priority to JP2019542804A priority patent/JP2019535306A/ja
Priority to BR112019008345A priority patent/BR112019008345A8/pt
Priority to CN201780080063.4A priority patent/CN110121508A/zh
Priority to EP17797084.5A priority patent/EP3532502A1/fr
Priority to KR1020197014634A priority patent/KR20190084264A/ko
Publication of WO2018077926A1 publication Critical patent/WO2018077926A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6817—Toxins
    • A61K47/6819—Plant toxins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/734—Complement-dependent cytotoxicity [CDC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present invention relates to antibodies (preferably monoclonal antibodies) binding to the CD160-TM isoform.
  • CD 160 has been initially identified as a GPI-anchored (CD 160-GPI) MHC-class I activating receptor mainly expressed on peripheral blood NK cells. It was additionally reported the identification of a CD 160 transmembrane isoform (CD160-TM) resulting from the alternative splicing of CD 160 gene. It was established that CD160-TM surface expression is highly restricted to NK cells and is activation-dependent (Giustiniani J et al. J Immunol. 2009 Jan 1;182(1):63-71). Indeed, CD160-TM is only expressed by activated NK cells, whereas CD 160-GPI is expressed by NK cells (activated or not) and by different subsets of T cells. In addition, it was provided evidences that CD160-TM represent a novel activating receptor, as assessed by the increased CD 107a NK cell surface mobilization observed upon its engagement (Giustiniani J et al. 2009).
  • CD 160 GPI-anchored isoform nor to the CD 160 soluble isoform that may result from the proteolytic cleavage of the CD 160-GPI isoform can thus be suitable, for example for amplifying NK cell activation and therefore effector functions of NK cells (cytotoxicity, cytokine secretion etc.) or for inducing depletion of CD160-TM expressing cells (in particular activated NK cells) in vivo.
  • using an antibody capable of binding to the CD160-TM isoform but not to the CD 160-GPI isoform will avoid any systemic toxicity such as cytokine storm risk.
  • WO2008/009711 describes antibody CL1-R2, an IgGl capable of binding CD160-GPI.
  • Giustiniani J. et al. describes a monoclonal antibody that binds to the CD160-TM isoform. However, this antibody also binds the soluble isoform of CD 160.
  • WO2008/155363 describes the production of polyclonal antibodies directed to CD 160- TM but not binding to the CD 160-GPI isoform. These antibodies were obtained by immunizing rabbits with a peptide (peptide 2) comprising amino acids 144-158 of CD160-TM (KQRQHLEF SHNNEGTL , SEQ ID NO: 32). In the present invention, the Inventors developed a novel antibody binding to the CD160-TM isoform, but not to the CD160-GPI or to the soluble CD 160 isoforms.
  • the present invention relates to human antibodies (preferably monoclonal antibodies) binding to the CD160-TM isoform.
  • the present invention is defined by the claims.
  • the present invention relates to a monoclonal antibody which binds to the extracellular domain of the CD160-TM isoform, wherein said antibody does not bind to the GPI-anchored isoform nor to the CD 160 soluble isoform, and wherein the epitope of said monoclonal antibody comprises at least one amino acid residue from amino acid residues 175 to 189 of SEQ ID NO: 1.
  • said epitope further comprises at least one amino acid residue from amino acid residues 62 to 85 of SEQ ID NO: 1.
  • the monoclonal antibody of the invention is a chimeric antibody, a humanized antibody or a human antibody.
  • the monoclonal antibody of the invention comprises a light chain comprising at least one of the following CDR: i) the VL-CDR1 as set forth in SEQ ID NO: 6 wherein Xn is Y or S and X12 is G or Y, ii) the VL-CDR2 as set forth in SEQ ID NO: 7 and iii) the VL-CDR3 as set forth in SEQ ID NO: 8 wherein X3 is S or Y, and/or a heavy chain comprising at least one of the following CDR i) the VH-CDR1 as set forth in SEQ ID NO: 9 wherein X 3 is S or Y, ii) the VH-CDR2 as set forth in SEQ ID NO: 10 wherein Xi is Y or G and X10 is N or S and iii) the VH-CDR3 as set forth in SEQ ID NO: 11.
  • the monoclonal antibody of the invention comprises a light chain comprising the following CDR: i) the VL-CDR1 as set forth in SEQ ID NO: 6 wherein X11 is Y or S and X12 is G or Y, ii) the VL-CDR2 as set forth in SEQ ID NO: 7 and iii) the VL-CDR3 as set forth in SEQ ID NO: 8 wherein X 3 is S or Y, and a heavy chain comprising the following CDR i) the VH-CDR1 as set forth in SEQ ID NO: 9 wherein X 3 is S or Y, ii) the VH-CDR2 as set forth in SEQ ID NO: 10 wherein Xi is Y or G and X10 is N or S and iii) the VH-CDR3 as set forth in SEQ ID NO: 11.
  • the monoclonal antibody of the invention comprises a light chain comprising the following CDR: i) VL-CDR1 : AGTSSDVGGYYGVS (SEQ ID NO: 20), ii) VL-CDR2: YDSYRPS (SEQ ID NO: 7) and iii) VL-CDR3: SSSTYYSTRV (SEQ ID NO: 24), and the heavy chain of the A12 antibody comprises the following CDR i) VH-CDR1 : NYSMN (SEQ ID NO: 26), ii) VH-CDR2: YIYGSSRYISYADFVKG (SEQ ID NO: 29) and iii) VH- CDR3: GMDV (SEQ ID NO: 11).
  • the monoclonal antibody of the invention comprises a light chain comprising the following CDR: i) VL-CDR1 : AGTSSDVGGYSYVS (SEQ ID NO: 23), ii) VL-CDR2: YDSYRPS (SEQ ID NO: 7) and iii) VL-CDR3: SSYTYYSTRV (SEQ ID NO: 25), and the heavy chain of the A12 antibody comprises the following CDR i) VH-CDR1 : NYYMN (SEQ ID NO: 27), ii) VH-CDR2: GIYGSSRYINYADFVKG (SEQ ID NO: 30) and iii) VH- CDR3: GMDV (SEQ ID NO: 11).
  • the monoclonal antibody of the invention comprises a heavy chain having at least 70% of identity with SEQ ID NO : 12 or SEQ ID NO : 14 and a light chain having at least 70 % of identity with SEQ ID NO: 13 or SEQ ID NO: 15. In one embodiment, the monoclonal antibody of the invention comprises a heavy chain identical to SEQ ID NO: 12 or SEQ ID NO: 14 and a light chain identical to SEQ ID NO: 13 or SEQ ID NO: 15.
  • the monoclonal antibody of the invention cross-competes for binding to the CD160-TM isoform with the antibody as described hereinabove.
  • the monoclonal antibody of the invention is conjugated to a cytotoxic moiety.
  • the present invention further relates to a fusion protein comprising a monoclonal antibody as described hereinabove.
  • the present invention further relates to a nucleic acid molecule which encodes a heavy chain or a light chain of the antibody as described hereinabove.
  • the nucleic acid molecule of the invention comprises a nucleic acid sequence having 70% of identity with SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 19.
  • the present invention further relates to a host cell which has been transfected, infected or transformed by the nucleic acid as described hereinabove.
  • the monoclonal antibody of the invention mediates antibody dependent cellular cytotoxicity, complement dependent cytotoxicity or antibody-dependent phagocytosis.
  • the present invention further relates to a monoclonal antibody as described hereinabove, for use in a method of treating a cancer wherein cancer cells express CD160-TM, preferably for treating a NK leukemia or a NK lymphoma, such as for example, extranodal and non-extranodal NK/T lymphomas; NK cell derived malignancies; and acute NK leukemia
  • the present invention further relates to a method of depleting a population of cells which express the CD160-TM isoform, a population of malignant NK cells which express the CD 160- TM isoform or a population of cells which express the epitope recognized by the A12 or B6 antibody in a subject in need thereof comprising delivering to the subject a therapeutically effective amount of the monoclonal antibody as described hereinabove.
  • the monoclonal antibody of the invention does not mediate antibody dependent cellular cytotoxicity, complement dependent cytotoxicity or antibody-dependent phagocytosis.
  • the present invention further relates to a monoclonal antibody as described hereinabove, for use in a method of treating a cancer, an infectious disease or an autoimmune and/or inflammatory disease.
  • the present invention further relates to a method of enhancing NK cell activities in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the antibody as described hereinabove.
  • the subject suffers from a cancer, an infectious disease or an autoimmune and/or inflammatory disease.
  • the present invention further relates to a method of enhancing NK cell antibody-dependent cellular cytotoxicity (ADCC) of an antibody in a subject in need thereof comprising administering to the subject the antibody in combination with the monoclonal antibody of the present invention.
  • ADCC NK cell antibody-dependent cellular cytotoxicity
  • the present invention further relates to a method for inhibiting CD160-TM binding to a ligand thereof, comprising contacting CD160-TM with a monoclonal antibody as described hereinabove.
  • the present invention further relates to a method of treating Paroxysmal Nocturnal Hemoglobinuria in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the monoclonal antibody as described hereinabove, preferably wherein said antibody is a Fab.
  • the present invention further relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody as described hereinabove and a pharmaceutically acceptable carrier.
  • nucleic acid bases In the context of the present invention, the following abbreviations for the commonly occurring nucleic acid bases are used. "A” refers to adenine, “C” refers to cytosine, “G” refers to guanine, “T” refers to thymine, and “U” refers to uracil.
  • the terms “a” and “an” refer to one or to more than one (i.e. , to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.
  • nucleic acid or “polynucleotide” refers to a polymer of nucleotides covalently linked by phosphodiester bonds, such as deoxyribonucleic acids (DNA) or ribonucleic acids (RNA), in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al, Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al, J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al, Mol. Cell. Probes 8:91-98 (1994)).
  • polypeptide refers to a compound comprised of amino acid residues covalently linked by peptide bonds.
  • a polypeptide is not limited to a specific length: it must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a polypeptide's sequence.
  • Peptides, oligopeptides, and proteins are included within the definition of polypeptide, and such terms may be used interchangeably herein unless specifically indicated otherwise.
  • the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types.
  • peptides refers to a linear polymer of amino acids linked together by peptide bonds, preferably having a chain length of less than about 50 amino acids residues; a "polypeptide” refers to a linear polymer of at least 50 amino acids linked together by peptide bonds; and a protein specifically refers to a functional entity formed of one or more peptides or polypeptides, optionally glycosylated, and optionally of non-polypeptides cofactors. This term also does exclude post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.
  • a polypeptide may be an entire protein, or a subsequence thereof.
  • Polypeptides include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others.
  • a polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof.
  • Particular polypeptides of interest in the context of this invention are amino acid subsequences comprising CDRs and being capable of binding an antigen.
  • a subject refers to a warm-blooded animal, preferably a mammal (including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc...), and more preferably a human.
  • a subject may be a "patient", i.e., a warm-blooded animal, more preferably a human, who/which is awaiting the receipt of, or is receiving medical care or was/is/will be the object of a medical procedure, or is monitored for the development of a disease.
  • the subject is an adult (for example a subject above the age of 18).
  • the subject is a child (for example a subject below the age of 18).
  • the subject is a male.
  • the subject is a female. DETAILED DESCRIPTION OF THE INVENTION:
  • the first object of the present invention relates to an antibody which binds to the CD 160- TM isoform but does not bind to the CD 160 GPI-anchored isoform.
  • the antibody of the invention binds to the extracellular domain of the CD160-TM isoform.
  • the antibody of the invention does not bind to the soluble CD 160 isoform.
  • the present invention refers to an antibody that binds to the extracellular domain of the CD160-TM isoform but does not bind to the CD 160 GPI- anchored isoform nor the soluble CD 160 isoform.
  • said antibody is a monoclonal antibody. Therefore, in one embodiment, the present invention refers to a monoclonal antibody that binds to the extracellular domain of the CD160-TM isoform but does not bind to the CD 160 GPI-anchored isoform nor the soluble CD 160 isoform.
  • said antibody is a polyclonal antibody.
  • antibody or “immunoglobulin” have the same meaning, and will be used equally in the present invention.
  • the term antibody encompasses not only whole antibody molecules, but also antibody fragments as well as variants (including derivatives) of antibodies and antibody fragments (e.g., Fab, Fab', F(ab') 2 or scFv. ..).
  • each heavy chain is linked to a light chain by a disulfide bond.
  • Each chain contains distinct sequence domains.
  • the light chain includes two domains, a variable domain (VL) and a constant domain (CL).
  • the heavy chain includes four domains, a variable domain (VH) and three constant domains (CHI, CH2 and CH3, collectively referred to as CH).
  • variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen.
  • the constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, trans-placental mobility, complement binding, and binding to Fc receptors (FcR).
  • the Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain.
  • the specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant.
  • Antibody combining sites are made up of residues that are primarily from the hypervariable or complementarity determining regions (CDRs).
  • Complementarity Determining Regions or CDRs refer to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site.
  • the light and heavy chains of an immunoglobulin each have three CDRs, designated VL-CDR1 , VL-CDR2, VL-CDR3 and VH-CDR1 , VH-CDR2, VH-CDR3, respectively.
  • An antigen-binding site therefore, typically includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region.
  • Framework Regions refer to amino acid sequences interposed between CDRs.
  • the residues in antibody variable domains are conventionally numbered according to a system devised by Kabat et al. This system is set forth in Kabat et al., 1987, in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA (hereafter "Kabat et al.”). This numbering system is used in the present specification. The Kabat residue designations do not always correspond directly with the linear numbering of the amino acid residues in SEQ ID sequences.
  • the actual linear amino acid sequence may contain fewer or additional amino acids than in the strict Kabat numbering corresponding to a shortening of, or insertion into, a structural component, whether framework or complementarity determining region (CDR), of the basic variable domain structure.
  • CDR complementarity determining region
  • the correct Kabat numbering of residues may be determined for a given antibody by alignment of residues of homology in the sequence of the antibody with a "standard" Kabat numbered sequence.
  • the CDRs of the heavy chain variable domain are located at residues 31- 35B (VH-CDR1), residues 50-65 (VH-CDR2) and residues 95-102 (VH-CDR3) according to the Kabat numbering system.
  • the CDRs of the light chain variable domain are located at residues 24-34 (VL-CDR1), residues 50-56 (VL-CDR2) and residues 89-97 (VL-CDR3) according to the Kabat numbering system.
  • an “intact” antibody is one which comprises an antigen-binding site as well as a CL and at least heavy chain constant domains, CHI , CH2 and CH3.
  • the constant domains may be native sequence constant domains (e.g. , human native sequence constant domains) or amino acid sequence variants thereof.
  • variable refers to the fact that certain segments of the V domains differ extensively in sequence among antibodies.
  • the V domain mediates antigen binding and defines specificity of a particular antibody for its particular antigen.
  • variability is not evenly distributed across the 1 10 to 130-amino acid span of the variable domains.
  • the V regions consist of relatively invariant stretches called framework regions (FRs) of 15-30 amino acids separated by shorter regions of extreme variability called “hypervariable regions” that are each 9-12 amino acids long.
  • FRs framework regions
  • hypervariable regions that are each 9-12 amino acids long.
  • the variable domains of native heavy and light chains each comprise four FRs, largely adopting a [beta] -sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the [beta]-sheet structure.
  • the hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).
  • the constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC).
  • the "variable region" or “variable domain” of an antibody refers to the amino-terminal domains of the heavy or light chain of the antibody.
  • the variable domain of the heavy chain may be referred to as "VH”.
  • the variable domain of the light chain may be referred to as "VL”.
  • hypervariable region when used herein refers to the amino acid residues of an antibody that are responsible for antigen binding.
  • the hypervariable region generally comprises amino acid residues from a "complementarity determining region" or "CDR" (e.g. , around about residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the VL, and around about 31- 35 (HI), 50-65 (H2) and 95-102 (H3) in the VH when numbered in accordance with the Kabat numbering system; Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).
  • CDR complementarity determining region
  • the antibody (preferably the monoclonal antibody) of the present invention is an antibody molecule selected from the group consisting of a whole antibody, a humanized antibody, a single chain antibody, a dimeric single chain antibody, a Fv, a scFv, a Fab, a F(ab)' 2 , a defucosylated antibody, a bi-specific antibody, a diabody, a triabody, a tetrabody.
  • said antibody is an antibody fragment selected from the group consisting of a unibody, a domain antibody, and a nanobody.
  • said antibody is an antibody mimetic selected from the group consisting of an affibody, an affilin, an affitin, an adnectin, an atrimer, an evasin, a DARPin, an anticalin, an avimer, a fynomer, a versabody and a duocalin.
  • a “nanobody” is well known in the art and refers to an antibody-derived therapeutic protein that contains the unique structural and functional properties of naturally-occurring heavy chain antibodies. These heavy chain antibodies contain a single variable domain (VHH) and two constant domains (CH2 and CH3).
  • VHH single variable domain
  • CH2 and CH3 constant domains
  • derived indicates a relationship between a first and a second molecule. It generally refers to structural similarity between the first molecule and the second molecule and does not connote or include a process or source limitation on a first molecule that is derived from a second molecule.
  • diabodies refers to small antibody fragments prepared by constructing sFv fragments with short linkers (about 5-10 residues) between the VH and VL domains such that inter-chain but not intra-chain pairing of the V domains is achieved, resulting in a bivalent fragment, i.e., fragment having two antigen binding sites.
  • Bispecific diabodies are heterodimers of two "crossover" sFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains.
  • Diabodies are described more fully in, for example, EP 0404097; WO 93/1 1 161 ; and Holliger et al, Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).
  • an “affibody” is well known in the art and refers to affinity proteins based on a 58 amino acid residue protein domain, derived from one of the IgG binding domain of staphylococcal protein A.
  • Anticalins are well known in the art and refer to an antibody mimetic technology, wherein the binding specificity is derived from lipocalins. Anticalins may also be formatted as dual targeting protein, called Duocalins.
  • a "Avimers” are well known in the art and refer to an antibody mimetic technology.
  • a “domain antibody” is well known in the art and refers to the smallest functional binding units of antibodies, corresponding to the variable regions of either the heavy or light chains of antibodies.
  • a "unibody” is well known in the art and refers to an antibody fragment lacking the hinge region of IgG4 antibodies. The deletion of the hinge region results in a molecule that is essentially half the size of traditional IgG4 antibodies and has a univalent binding region rather than the bivalent biding region of IgG4 antibodies.
  • DARPins Designed Ankyrin Repeat Proteins
  • DRP designed repeat protein
  • antibody fragment refers to at least one portion of an intact antibody, preferably the antigen binding region or variable region of the intact antibody, that retains the ability to specifically interact with (e.g., by binding, steric hindrance, stabilizing/destabilizing, spatial distribution) an epitope of an antigen.
  • antibody fragments include, but are not limited to, Fab, Fab', F(ab') 2 , Fv fragments, single chain antibody molecules, in particular scFv antibody fragments, disulfide-linked Fvs (sdFv), a Fd fragment consisting of the VH and CHI domains, linear antibodies, single domain antibodies such as, for example, sdAb (either VL or VH), camelid VHH domains, multi-specific antibodies formed from antibody fragments such as, for example, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, and an isolated CDR or other epitope binding fragments of an antibody.
  • An antigen binding fragment can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, Nature Biotechnology 23 : 1 126-1 136, 2005).
  • Antigen binding fragments can also be grafted into scaffolds based on polypeptides such as a fibronectin type III (see U.S. Patent No.: 6,703,199, which describes fibronectin polypeptide minibodies). Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, and a residual "Fc" fragment, a designation reflecting the ability to crystallize readily.
  • a "functional fragment or analog of an antibody” is a compound having qualitative biological activity in common with a full-length antibody.
  • a functional fragment or analog of an anti-IgE antibody is one that can bind to an IgE immunoglobulin in such a manner so as to prevent or substantially reduce the ability of such molecule from having the ability to bind to the high affinity receptor, Fc[epsilon]RI.
  • the "Fc” fragment of an antibody comprises the carboxy-terminal portions of both H chains held together by disulfides.
  • the effector functions of antibodies are determined by sequences in the Fc region, which region is also the part recognized by Fc receptors (FcR) found on certain types of cells.
  • Fv is the minimum antibody fragment that contains a complete antigen-recognition and -binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (three loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
  • Fragments and derivatives of antibodies of this invention can be produced by techniques that are known in the art. "Fragments” comprise a portion of the intact antibody, generally the antigen binding site or variable region.
  • antibody fragments include Fab, Fab', Fab'-SH, F(ab')2, and Fv fragments; diabodies; any antibody fragment that is a polypeptide having a primary structure consisting of one uninterrupted sequence of contiguous amino acid residues (referred to herein as a "single-chain antibody fragment” or "single chain polypeptide"), including without limitation (1) single - chain Fv molecules (2) single chain polypeptides containing only one light chain variable domain, or a fragment thereof that contains the three CDRs of the light chain variable domain, without an associated heavy chain moiety and (3) single chain polypeptides containing only one heavy chain variable region, or a fragment thereof containing the three CDRs of the heavy chain variable region, without an associated light chain moiety; and multispecific antibodies formed from antibody fragments. Fragments of the present antibodies can be obtained using standard methods.
  • Fab or F(ab') 2 fragments may be produced by protease digestion of the isolated antibodies, according to conventional techniques. It will be appreciated that immunoreactive fragments can be modified using known methods, for example to slow clearance in vivo and obtain a more desirable pharmacokinetic profile the fragment may be modified with polyethylene glycol (PEG). Methods for coupling and site-specifically conjugating PEG to a Fab' fragment are described in, for example, Leong et al., Cytokines 16 (3): 106-119 (2001) and Delgado et al, Br. J. Cancer 5 73 (2): 175- 182 (1996), the disclosures of which are incorporated herein by reference.
  • PEG polyethylene glycol
  • the antibody (preferably the monoclonal antibody) of the invention is isolated.
  • an "isolated antibody” is one that has been separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that may interfere with diagnostic or therapeutic uses of the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous components.
  • the antibody is purified: (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator; or (3) to homogeneity as shown by SDS-PAGE under reducing or non-reducing conditions and using Coomassie blue or, preferably, silver staining.
  • Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
  • CD 160 has its general meaning in the art and refers to CD 160 molecule.
  • CD 160 gene was found to be located on human chromosome 1, and the corresponding protein was originally characterized as a glycosylphosphatidylinositol (GPI)- anchored cell surface molecule.
  • GPI glycosylphosphatidylinositol
  • CD160-GPI is expressed by intestinal intraepithelial T lymphocytes and by a minor subset of circulating lymphocytes including NK cells, TCRy5 and cytotoxic effector CD8 bright CD28 ⁇ T lymphocytes (ANUMANTHAN et al, 1998, J Immunol; 161 :2780-2790; MAIZA et al, J. Exp. Med., vol. 178, p: 1121-1126, 1993).
  • the CD160 transmembrane isoform (“CD160-TM") is described in Giustiniani J et al. (J Immunol.
  • CD160-TM The extracellular domain of the CD160-TM isoform may be defined by the amino acid sequence ranging from the amino acid residue at position 26 to the amino acid residue at position 189 in SEQ ID NO: 1.
  • CD160-GPI The CD 160 GPI-anchored isoform
  • CD160-GPI is described in Nikolova M. et al. (Int Immunol. 2002 May;14(5):445-51.) as well as in the international patent application WO2006015886 and is characterized by the amino acid sequence as set forth in SEQ ID NO: 2 fused to a GPI anchor at the C terminus end.
  • the CD 160 soluble isoform is described in Giustiniani J. et al. (J Immunol. 2007 Feb 1 ; 178(3): 1293-300) and is characterized by the amino acid sequence as set forth in SEQ ID NO: 3.
  • amino acids 1-25 correspond to a signal peptide, and may consequently be absent from the expressed protein.
  • SEQ ID NO: 3 CD 160 soluble isoform
  • binding refers to a direct association between two molecules, due to, for example, covalent, electrostatic, hydrophobic, and ionic and/or hydrogen-bond interactions, including interactions such as salt bridges and water bridges.
  • binding in the context of the binding of an antibody to a predetermined antigen or epitope typically is a binding with an affinity corresponding to a KD of about 10 "7 M or less, such as about 10 "8 M or less, such as about 10 "9 M or less, about 10 "10 M or less, or about 10 "11 M or even less.
  • Methods for measuring the KD of an antibody include, without limitation, surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument using a soluble form of the antigen as the ligand and the antibody as the analyte.
  • SPR surface plasmon resonance
  • BIACORE® GE Healthcare, Piscaataway, NJ
  • Affinities of antibodies can be readily determined using other conventional techniques, for example, those described by Scatchard et al, (Ann. N.Y. Acad. Sci. USA 51 :660 (1949)).
  • Binding properties of an antibody to antigens, cells or tissues may generally be determined and assessed using immunodetection methods including, for example, immunofluorescence-based assays, such as immunohistochemistry (IHC) and/or fluorescence-activated cell sorting (FACS).
  • immunofluorescence-based assays such as immunohistochemistry (IHC) and/or fluorescence-activated cell sorting (FACS).
  • an antibody binds to the predetermined antigen with an affinity corresponding to a KD that is at least ten- fold lower, such as at least 100-fold lower, for instance at least 1 ,000-fold lower, such as at least 10,000-fold lower, for instance at least 100,000-fold lower than its KD for binding to a non-specific antigen (e.g. , BSA, casein), which is not identical or closely related to the predetermined antigen.
  • a non-specific antigen e.g. , BSA, casein
  • the KD of the antibody When the KD of the antibody is very low (that is, the antibody has a high affinity), then the KD with which it binds the antigen is typically at least 10,000-fold lower than its KD for a non-specific antigen.
  • An antibody is said to essentially not bind an antigen or epitope if such binding is either not detectable (using, for example, plasmon resonance (SPR) technology in a BIAcore 3000 instrument using a soluble form of the antigen as the ligand and the antibody as the analyte), or is 100 fold, 500 fold, 1000 fold or more than 1000 fold less than the binding detected by that antibody and an antigen or epitope having a different chemical structure or amino acid sequence.
  • SPR plasmon resonance
  • the term "specificity" refers to the ability of an antibody to detectably bind an epitope presented on an antigen, such as a CD160-TM, while having relatively little detectable reactivity with non-CD 160-TM proteins such as the CD 160 GPI-anchored isoform and the CD 160 soluble isoform. Specificity can be relatively determined by binding or competitive binding assays, using, e.g. , Biacore instruments, as described elsewhere herein.
  • Specificity can be exhibited by, e.g., an about 10: 1 , about 20: 1 , about 50: 1 , about 100: 1 , 10.000: 1 or greater ratio of affinity/avidity in binding to the specific antigen versus nonspecific binding to other irrelevant molecules (in this case the specific antigen is a CD 160-TM polypeptide).
  • affinity means the strength of the binding of an antibody to an epitope.
  • the affinity of an antibody is given by the dissociation constant Kd, defined as [Ab] x [Ag] / [Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody- antigen complex, [Ab] is the molar concentration of the unbound antibody and [Ag] is the molar concentration of the unbound antigen.
  • Kd dissociation constant
  • Ka is defined by 1/Kd.
  • the antibody of the invention binds to an epitope comprising at least one amino acid residue from amino acid residues 175 to 189 of SEQ ID NO: 1, or from a sequence sharing at least 60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% of identity over amino acid residues 175 to 189 of SEQ ID NO : 1.
  • Amino acid residues 175 to 189 of SEQ ID NO: 1 correspond to the sequence SEQ ID NO: 5 (LVALQGMSKRAVSTP).
  • epitope refers to a specific arrangement of amino acids located on a protein or proteins to which an antibody binds. Epitopes often consist of a chemically active surface grouping of molecules such as amino acids or sugar side chains, and have specific three dimensional structural characteristics as well as specific charge characteristics. Epitopes can be linear or conformational, i.e., involving two or more sequences of amino acids in various regions of the antigen that may not necessarily be contiguous.
  • the antibody of the invention binds to an epitope comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues from amino acid residues 175 to 189 of SEQ ID NO: 1, or from a sequence sharing at least 60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% of identity over amino acid residues 175 to 189 of SEQ ID NO: 1.
  • the antibody of the invention binds to an epitope comprising the amino acid sequence as set forth in SEQ ID NO: 5 (LVALQGMSKRAVSTP) or an amino acid sequence sharing at least 60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% of identity over SEQ ID NO: 5.
  • the antibody of the invention binds to an epitope comprising at least one amino acid residue from amino acid residues 62 to 85 of SEQ ID NO: 1 , or from a sequence sharing at least 60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% of identity over amino acid residues 62 to 85 of SEQ ID NO: 1.
  • Amino acid residues 62 to 85 of SEQ ID NO: 1 correspond to the sequence SEQ ID NO: 4 (KDRSGDCSPETSLKQLRLK DPGI).
  • the antibody of the invention binds to an epitope comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 amino acid residues from amino acid residues 62 to 85 of SEQ ID NO: 1, or from a sequence sharing at least 60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% of identity over amino acid residues 62 to 85 of SEQ ID NO: 1.
  • the antibody of the invention binds to an epitope comprising the amino acid sequence as set forth in SEQ ID NO: 4 (KDRSGDCSPETSLKQLRLKRDPGI) or an amino acid sequence sharing at least 60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% of identity over SEQ ID NO: 4.
  • the antibody of the invention binds to a conformational epitope.
  • the antibody of the invention binds to a conformational epitope comprising:
  • At least one amino acid residue from amino acid residues 62 to 85 of SEQ ID NO: 1, or from a sequence sharing at least 60%, 70%, 75%, 80%, 90%, 95%,
  • the antibody of the invention binds to a conformational epitope comprising:
  • amino acid residues from amino acid residues 175 to 189 of SEQ ID NO: 1, or from a sequence sharing at least 60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% of identity over amino acid residues 175 to 189 of SEQ ID NO: 1, and
  • amino acid residues from amino acid residues 62 to 85 of SEQ ID NO: 1, or from a sequence sharing at least 60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%) of identity over amino acid residues 62 to 85 of SEQ ID NO: 1.
  • the antibody of the invention binds to a conformational epitope comprising: the amino acid sequence as set forth in SEQ ID NO: 5 (LVALQGMSKRAVSTP) or an amino acid sequence sharing at least 60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% of identity over SEQ ID NO: 5, and
  • amino acid residues 62 to 85 of SEQ ID NO: 1 or from a sequence sharing at least 60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% of identity over amino acid residues 62 to 85 of SEQ ID NO: 1.
  • the antibody of the invention binds to a conformational epitope comprising or consisting of:
  • amino acid sequence as set forth in SEQ ID NO: 5 (LVALQGMSKRAVSTP) or an amino acid sequence sharing at least 60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% of identity over SEQ ID NO: 5, and
  • KDRSGDCSPETSLKQLRLKRDPGI amino acid sequence sharing at least 60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% of identity over SEQ ID NO: 4.
  • the monoclonal antibody of the present invention binds to the extracellular domain of the CD160-TM isoform in the amino acid sequence as set forth in SEQ ID NO: 4 (KDRSGDCSPETSLKQLRLKRDPGI) and in the amino acid sequence as set forth in SEQ ID NO: 5 (LVALQGMSKRAVSTP).
  • monoclonal antibody refers to a preparation of antibody molecules of single molecular composition.
  • a monoclonal antibody is obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprised in the population are identical except for possible naturally occurring mutations that may be present in minor amounts.
  • a monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.
  • Monoclonal antibodies may be generated using the method of Kohler and Milstein (Nature, 256:495, 1975). To prepare monoclonal antibodies useful in the invention, a mouse or other appropriate host animal is immunized at suitable intervals (e.g.
  • Suitable immunologic adjuvants include Freund's complete adjuvant, Freund's incomplete adjuvant, alum, Ribi adjuvant, Hunter's Titermax, saponin adjuvants such as QS21 or Quil A, or CpG-containing immunostimulatory oligonucleotides. Other suitable adjuvants are well-known in the field.
  • the animals may be immunized by subcutaneous, intraperitoneal, intramuscular, intravenous, intranasal or other routes.
  • a given animal may be immunized with multiple forms of the antigen by multiple routes.
  • the modifier "monoclonal" is not to be construed as requiring production of the antibody by any particular method.
  • a monoclonal antibody may also be prepared by the hybridoma methodology first described by Kohler et al., Nature, 256:495 (1975), or may be made using recombinant DNA methods in bacterial, eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567).
  • a "monoclonal antibody” may also be isolated from phage antibody libraries using the techniques described in Clackson et al, Nature, 352:624-628 (1991) and Marks et al, J. Mol. Biol, 222:581-597 (1991), for example.
  • the monoclonal antibody of the invention is a chimeric antibody, in particular a chimeric mouse/human antibody.
  • chimeric antibody refers to an antibody which comprises a VH domain and a VL domain of a non-human antibody, and a CH domain and a CL domain of a human antibody.
  • a "chimeric antibody” is an antibody molecule in which (a) the constant region (i.e., the heavy and/or light chain), or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g. , an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.
  • Chimeric antibodies also include primatized and in particular humanized antibodies.
  • chimeric antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. For further details, see Jones et al., Nature 321 :522-525 (1986); Riechmann et al, Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593- 596 (1992). (see U.S. Pat. No. 4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. USA, 81 :6851-6855 (1984)).
  • the monoclonal antibody of the invention is a humanized antibody.
  • the variable domain comprises human acceptor frameworks regions, and optionally human constant domain where present, and non- human donor CDRs, such as mouse CDRs.
  • the term "humanized antibody” refers to an antibody having variable region framework and constant regions from a human antibody but retains the CDRs of a previous non-human antibody.
  • a humanized antibody contains minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies and antibody fragments thereof may be human immunoglobulins (recipient antibody or antibody fragment) in which residues from a complementary-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity.
  • CDR complementary-determining region
  • donor antibody such as mouse, rat or rabbit having the desired specificity, affinity, and capacity.
  • Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • a humanized antibody/antibody fragment can comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. Such antibodies are designed to maintain the binding specificity of the non-human antibody from which the binding regions are derived, but to avoid an immune reaction against the non-human antibody.
  • the humanized antibody or antibody fragment thereof will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or a significant portion of the FR regions are those of a human immunoglobulin sequence.
  • the humanized antibody or antibody fragment can also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • the monoclonal antibody is a human monoclonal antibody.
  • human monoclonal antibody is intended to include antibodies having variable and constant regions derived from human immunoglobulin sequences.
  • the human antibodies of the present invention may include amino acid residues not encoded by human immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
  • the term "human monoclonal antibody”, as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • the antibody of the invention comprises a light chain comprising at least one or at least two of the following CDRs:
  • VL-CDR2 YDSYRPS (SEQ ID NO: 7);
  • the antibody of the invention comprises a light chain comprising the following CDRs:
  • VL-CDR2 YDSYRPS (SEQ ID NO: 7);
  • VL-CDR1 has a sequence selected from AGTSSDVGGYYGVS (SEQ ID NO: 20), AGTSSDVGGYYYVS (SEQ ID NO: 21), AGTSSDVGGYSGVS (SEQ ID NO: 22), and AGTSSDVGGYSYVS (SEQ ID NO: 23).
  • VL-CDR3 is selected from SSSTYYSTRV (SEQ ID NO: 24) and SSYTYYSTRV (SEQ ID NO: 25).
  • the antibody of the invention comprises a light chain comprising the three following CDRs:
  • VL-CDR2 YDSYRPS (SEQ ID NO: 7);
  • the antibody of the invention comprises a light chain comprising the three following CDRs:
  • VL-CDR2 YDSYRPS (SEQ ID NO: 7);
  • the antibody of the invention comprises a light chain comprising the three following CDRs:
  • VL-CDR2 YDSYRPS (SEQ ID NO: 7);
  • the antibody of the invention comprises a light chain comprising the three following CDRs: - VL-CDR1 : AGTSSDVGGYYYVS (SEQ ID NO: 21);
  • VL-CDR2 YDSYRPS (SEQ ID NO: 7);
  • the antibody of the invention comprises a light chain comprising the three following CDRs:
  • VL-CDR2 YDSYRPS (SEQ ID NO: 7);
  • the antibody of the invention comprises a light chain comprising the three following CDRs :
  • VL-CDR2 YDSYRPS (SEQ ID NO: 7);
  • the antibody of the invention comprises a light chain comprising the three following CDRs:
  • VL-CDR2 YDSYRPS (SEQ ID NO: 7);
  • the antibody of the invention comprises a light chain comprising the three following CDRs:
  • VL-CDR2 YDSYRPS (SEQ ID NO: 7);
  • the antibody of the invention comprises a heavy chain comprising at least one or at least two of the following CDRs:
  • the antibody of the invention comprises a heavy chain comprising the following CDRs:
  • VH-CDR1 NY-X 3 -MN, wherein X 3 is S or Y (SEQ ID NO: 9) - VH-CDR2: Xi-IYGSSRYI-Xio-YADFVKG, wherein Xi is Y or G and Xio is N or S (SEQ ID NO: 10); and
  • VH-CDRl has a sequence selected from NYSMN (SEQ ID NO: 26) and NYYMN (SEQ ID NO: 27).
  • VH-CDR2 has a sequence selected from YIYGSSRYINYADFVKG (SEQ ID NO: 28), YIYGSSRYISYADFVKG (SEQ ID NO: 29), GIYGSSRYINYADFVKG (SEQ ID NO: 30) and GIYGSSRYISYADFVKG (SEQ ID NO: 31).
  • the antibody of the invention comprises a heavy chain comprising the three following CDRs:
  • VH-CDR2 YIYGSSRYINYADFVKG (SEQ ID NO: 28);
  • the antibody of the invention comprises a heavy chain comprising the three following CDRs:
  • VH-CDR2 YIYGSSRYISYADFVKG (SEQ ID NO: 29);
  • the antibody of the invention comprises a heavy chain comprising the three following CDRs:
  • VH-CDR2 GIYGSSRYINYADFVKG (SEQ ID NO: 30);
  • the antibody of the invention comprises a heavy chain comprising the three following CDRs:
  • VH-CDR2 GIYGSSRYISYADFVKG (SEQ ID NO: 31);
  • the antibody of the invention comprises a heavy chain comprising the three following CDRs:
  • VH-CDR2 YIYGSSRYINYADFVKG (SEQ ID NO: 28);
  • the antibody of the invention comprises a heavy chain comprising the three following CDRs:
  • VH-CDR2 YIYGSSRYISYADFVKG (SEQ ID NO: 29);
  • the antibody of the invention comprises a heavy chain comprising the three following CDRs:
  • VH-CDR2 GI YGS SRYINYADF VKG (SEQ ID NO: 30);
  • the antibody of the invention comprises a heavy chain comprising the three following CDRs:
  • VH-CDR2 GIYGSSRYISYADFVKG (SEQ ID NO: 31);
  • the monoclonal antibody of the present invention comprises a light chain comprising i) the VL-CDR1 as set forth in SEQ ID NO: 6 wherein Xn is Y or S and Xi2 is G or Y, ii) the VL-CDR2 as set forth in SEQ ID NO: 7 and iii) the VL-CDR3 as set forth in SEQ ID NO: 8 wherein X 3 is S or Y, and a heavy chain comprising i) the VH-CDRl as set forth in SEQ ID NO: 9 wherein X 3 is S or Y, ii) the VH-CDR2 as set forth in SEQ ID NO: 10 wherein Xi is Y or G and Xio is N or S and iii) the VH-CDR3 as set forth in SEQ ID NO: 11.
  • any of the CDRs 1 , 2 and 3 of the heavy and light chains may be characterized as having an amino acid sequence that shares at least 60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% of identity with the particular CDR or sets of CDRs listed in the corresponding SEQ ID NO.
  • the monoclonal antibody of the present invention comprises a light chain comprising i) the VL-CDR1 of A12, ii) the VL-CDR2 of A12 and iii) the VL-CDR3 of A12, and a heavy chain comprising i) the VH-CDRl of A12, ii) the VH-CDR2 of A12 and iii) the VH-CDR3 of A12.
  • the VH region of the A12 antibody consists of the sequence of SEQ ID NO: 12. Accordingly, the VH-CDRl of A12 is defined by the sequence ranging from the amino acid residue at position 31 to the amino acid residue at position 35 in SEQ ID NO: 12. Accordingly, the VH-CDR2 of A12 is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 66 in SEQ ID NO:
  • VH-CDR3 of A12 is defined by the sequence ranging from the amino acid residue at position 103 to the amino acid residue at position 106 in SEQ ID NO: 12.
  • SEQ ID NO: 12 VH region of A12 antibody FR1 -CDR1 -FR2-CDR2-FR3-CDR3-FR4 EVQLVESGGSLVKPGGSLRLSCAASGFTFS YSMNWVRQAPGKGLEWISYIYGSSRYISYADFV KGRFTISRDNATNSLYLQMNSLRAEDTAVYYCVRSYYGGMDVWGRGTLVTVSS
  • the VL region of the A12 antibody consists of the sequence of SEQ ID NO: 13. Accordingly, the VL-CDRl of A12 is defined by the sequence ranging from the amino acid residue at position 23 to the amino acid residue at position 36 in SEQ ID NO: 13. Accordingly, the VL-CDR2 of A12 is defined by the sequence ranging from the amino acid residue at position 52 to the amino acid residue at position 58 in SEQ ID NO:
  • VL-CDR3 of A12 is defined by the sequence ranging from the amino acid residue at position 91 to the amino acid residue at position 100 in SEQ ID NO: 13.
  • SEQ ID NO: 13 VL region of A12 antibody FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 QSVLTQPASVSGSPGQSITISCAGTSSDVGGYYGVSWYQQHPGKAPKLMIYYDSYRPSGVSNRF SGSKSGNT ASLTISGLQAEDE ADYYC S S ST YYSTRVFGGGTKLEK
  • the light chain of the A12 antibody comprises the following CDR: i) VL-CDRl : SEQ ID NO: 20, ii) VL-CDR2: SEQ ID NO: 7 and iii) VL-CDR3: SEQ ID NO: 24, and the heavy chain of the A12 antibody comprises the following CDR i) VH-CDRl : SEQ ID NO: 26, ii) VH-CDR2: SEQ ID NO: 29 and iii) VH-CDR3: SEQ ID NO: 11.
  • the monoclonal antibody of the present invention comprises a light chain comprising i) the VL-CDRl of B6, ii) the VL-CDR2 of B6 and iii) the VL-CDR3 of B6, and a heavy chain comprising i) the VH-CDRl of B6, ii) the VH-CDR2 of B6 and iii) the VH-CDR3 of B6.
  • the VH region of the B6 antibody consists of the sequence of SEQ ID NO: 14. Accordingly, the VH-CDRl of B6 is defined by the sequence ranging from the amino acid residue at position 31 to the amino acid residue at position 35 in SEQ ID NO: 14. Accordingly, the VH-CDR2 of B6 is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 66 in SEQ ID NO:
  • VH-CDR3 of B6 is defined by the sequence ranging from the amino acid residue at position 103 to the amino acid residue at position 106 in SEQ ID NO: 14.
  • SEQ ID NO: 14 VH region of B6 antibody FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 EVQLVESGGSLVKPGGSLRLSCAASGFTFSNYYMNWVRQAPGKGLEWISGIYGSSRYINYADF VKGRFTISRDNATNSLYLQMNSLRAEDTAVYYCVRSYYGGMDVWGRGTLVTVSS
  • the VL region of the B6 antibody consists of the sequence of SEQ ID NO: 15. Accordingly, the VL-CDRl of B6 is defined by the sequence ranging from the amino acid residue at position 23 to the amino acid residue at position 36 in SEQ ID NO: 15. Accordingly, the VL-CDR2 of B6 is defined by the sequence ranging from the amino acid residue at position 52 to the amino acid residue at position 58 in SEQ ID NO: 15. Accordingly, the VL-CDR3 of B6 is defined by the sequence ranging from the amino acid residue at position 91 to the amino acid residue at position 100 in SEQ ID NO: 15.
  • SEQ ID NO: 15 VL region of B6 antibody FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 QSVLTQPASVSGSPGQSITISCAGTSSDVGGYSYVSWYQQHPGKAPKLMlYYDSYRPSGVSNRF SGSKSGNTASLTISGLQAEDEADYYCSSYTYYSTRVFGGGTKLEK
  • the light chain of the B6 antibody comprises the following CDR: i) VL-CDRl : SEQ ID NO: 23, ii) VL-CDR2: SEQ ID NO: 7 and iii) VL-CDR3: SEQ ID NO: 25, and the heavy chain of the B6 antibody comprises the following CDR i) VH-CDR1 : SEQ ID NO: 27, ii) VH-CDR2: SEQ ID NO: 30 and iii) VH-CDR3: SEQ ID NO: 11.
  • the human monoclonal antibody of the present invention is an antibody comprising a heavy chain having at least 70% of identity with SEQ ID NO: 12 or SEQ ID NO: 14.
  • the human monoclonal antibody of the present invention is an antibody comprising a light chain having at least 70% of identity with SEQ ID NO: 13 or SEQ ID NO: 15.
  • the human monoclonal antibody of the present invention is an antibody comprising a heavy chain having at least 70% of identity with SEQ ID NO: 12 or SEQ ID NO: 14 and a light chain having at least 70% of identity with SEQ ID NO: 13 or SEQ ID NO: 15.
  • a first amino acid sequence having at least 70% of identity with a second amino acid sequence means that the first sequence has 70; 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% of identity with the second amino acid sequence.
  • Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar are the two sequences.
  • ALIGN Myers and Miller, CABIOS 4: 11-17, 1989
  • LFASTA Nearson and Lipman, 1988
  • ALIGN compares entire sequences against one another
  • LFASTA compares regions of local similarity.
  • the Blast 2 sequences function can be employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1).
  • the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties).
  • the BLAST sequence comparison system is available, for instance, from the NCBI web site; see also Altschul et al, J. Mol. Biol, 215:403-410, 1990; Gish. & States, Nature Genet., 3:266-272, 1993; Madden et al. Meth. EnzymoL, 266:131-141, 1996; Altschul et al, Nucleic Acids Res., 25:3389-3402, 1997; and Zhang & Madden, Genome Res., 7:649-656, 1997.
  • the human monoclonal antibody of the present invention is an antibody comprising a heavy chain which is identical to SEQ ID NO: 12 or SEQ ID NO: 14.
  • the human monoclonal antibody of the present invention is an antibody comprising a light chain identical to SEQ ID NO: 13 or SEQ ID NO: 15.
  • the human monoclonal antibody of the present invention is an antibody comprising a heavy chain identical to SEQ ID NO: 12 or SEQ ID NO: 14 and a light chain identical to SEQ ID NO: 13 or SEQ ID NO: 15.
  • the human monoclonal antibody of the present invention is an antibody comprising a heavy chain identical to SEQ ID NO: 12 and a light chain identical to SEQ ID NO: 13. In one embodiment, the human monoclonal antibody of the present invention is an antibody comprising a heavy chain identical to SEQ ID NO: 12 and a light chain identical to SEQ ID NO: 15. In one embodiment, the human monoclonal antibody of the present invention is an antibody comprising a heavy chain identical to SEQ ID NO: 14 and a light chain identical to SEQ ID NO: 13. In one embodiment, the human monoclonal antibody of the present invention is an antibody comprising a heavy chain identical to SEQ ID NO: 14 and a light chain identical to SEQ ID NO : 15.
  • the heavy chain and/or the light chain of the antibody of the invention comprises conservative sequence modifications as compared to the SEQ ID NO defined hereinabove, for example 1 to 10 conservative sequence modifications.
  • conservative sequence modifications refers to amino acid modifications that do not significantly affect or alter the biologic function of the protein containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into a protein by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • a “conservative substitution” is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged.
  • Amino acid substitutions are generally therefore based on the relative similarity of the amino acid side- chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
  • Amino acid substitutions may further be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues.
  • negatively charged amino acids include aspartic acid and glutamic acid
  • positively charged amino acids include lysine and arginine
  • amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine.
  • amino acids that may represent conservative changes include: (1) ala, pro, gly, glu, asp, gin, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his.
  • amino acids with basic side chains e.g. , lysine, arginine, histidine
  • acidic side chains e.g. , aspartic acid, glutamic acid
  • uncharged polar side chains e.g.
  • glycine asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan
  • nonpolar side chains e.g. , alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine
  • beta-branched side chains e.g. , threonine, valine, isoleucine
  • aromatic side chains e.g. , tyrosine, phenylalanine, tryptophan, histidine.
  • the monoclonal antibody of the present invention is selected from the group of Fab, F(ab') 2 , Fab' and scFv.
  • Fab denotes an antibody fragment having a molecular weight of about 50,000 and antigen binding activity, in which about a half of the N-terminal side of H chain and the entire L chain, among fragments obtained by treating IgG with a protease, papaine, are bound together through a disulfide bond.
  • F(ab') 2 refers to an antibody fragment having a molecular weight of about 100,000 and antigen binding activity, which is slightly larger than the Fab bound via a disulfide bond of the hinge region, among fragments obtained by treating IgG with a protease, pepsin.
  • Fab' refers to an antibody fragment having a molecular weight of about 50,000 and antigen binding activity, which is obtained by cutting a disulfide bond of the hinge region of the F(ab') 2 .
  • Fab'- SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group.
  • a single chain Fv (“scFv”) polypeptide is a covalently linked VH: :VL heterodimer which is usually expressed from a gene fusion including VH and VL encoding genes linked by a peptide-encoding linker.
  • the scFv fragment of the invention includes CDRs that are held in appropriate conformation, preferably by using gene recombination techniques.
  • the monoclonal antibody of the present invention cross-competes for binding to the CD160-TM isoform with the monoclonal antibody which comprises a light chain comprising i) the VL-CDR1 as set forth in SEQ ID NO: 6 wherein Xn is Y or S and X12 is G or Y, ii) the VL-CDR2 as set forth in SEQ ID NO: 7 and iii) the VL-CDR3 as set forth in SEQ ID NO: 8 wherein X3 is S or Y, and a heavy chain comprising i) the VH-CDR1 as set forth in SEQ ID NO: 9 wherein X 3 is S or Y, ii) the VH-CDR2 as set forth in SEQ ID NO: 10 wherein Xi is Y or G and X10 is N or S (SEQ ID NO: 10) and iii) the VH-CDR3 as set forth in SEQ ID NO: 1 1.
  • the monoclonal antibody of the present invention cross-competes for binding to the CD160-TM isoform with the monoclonal antibody which comprises the CDRs of A12 as defined above. In some embodiments, the monoclonal antibody of the present invention cross-competes for binding to the CD160-TM isoform with the monoclonal antibody which comprises CDRs of B6 as defined above.
  • cross-competes refers to monoclonal antibodies which share the ability to bind to a specific region of an antigen.
  • the monoclonal antibody that "cross-competes” has the ability to interfere with the binding of another monoclonal antibody for the antigen in a standard competitive binding assay.
  • Such a monoclonal antibody may, according to non-limiting theory, bind to the same or a related or nearby (e.g. , a structurally similar or spatially proximal) epitope as the antibody with which it competes.
  • Cross- competition is present if antibody A reduces binding of antibody B at least by 60%, specifically at least by 70% and more specifically at least by 80% and vice versa in comparison to the positive control which lacks one of said antibodies.
  • the cross-competing antibody as above described retain the activity of the monoclonal antibody which comprises a light chain comprising i) the VL-CDR1 as set forth in SEQ ID NO: 6 wherein Xn is Y or S and X12 is G or Y, ii) the VL- CDR2 as set forth in SEQ ID NO: 7 and iii) the VL-CDR3 as set forth in SEQ ID NO: 8 wherein X3 is S or Y, and a heavy chain comprising i) the VH-CDR1 as set forth in SEQ ID NO: 9 wherein X 3 is S or Y, ii) the VH-CDR2 as set forth in SEQ ID NO: 10 wherein Xi is Y or G and X10 is N or S and iii) the VH-CDR3 as set forth in SEQ ID NO: 1 1.
  • the cross- competing antibody retains the activity of the A12 or B6 antibody.
  • Any assay well known in the art would be suitable for identifying whether the cross-competing antibody retains the desired activity.
  • the assay described in EXAMPLE 4 that consist in determining the ability of increasing degranulation activity of the antibody would be suitable for determining whether the antibody retains the ability of increasing the NK cells activities, in particular NK cell killing activities.
  • the monoclonal antibody of the present invention does not cross-compete with the CL1-R2 antibody for binding to the CD 160 GPI-anchored isoform.
  • the CL1-R2 antibody does not cross-compete with the monoclonal antibody of the present invention for binding to the CD160-TM isoform.
  • CL1-R2 antibody is obtainable by the hybridoma deposited at the Collection Nationale de Cultures de Microorganismes C.N. CM. Institut Pasteur in accordance with the terms of the Budapest Treaty on April 28th, 2004 (C.N. CM. Institut Pasteur 25, rue du Dondel Roux F-75724 Paris Cedex 15 France).
  • the deposited hybridoma has CNCM deposit number 1-3204.
  • the monoclonal antibody of the present invention does not cross-compete with the BY55 antibody for binding to the CD 160 GPI- anchored isoform.
  • the BY55 antibody does not cross-compete with the monoclonal antibody of the present invention for binding to the CD160-TM isoform.
  • BY55 may be obtained, for example, from Abeam (reference number ab81388) and from ThermoFisher Scientific (reference number 12-1609-42).
  • the invention also provides an antibody that binds essentially the same epitope as A12 or B6 antibodies as described hereinabove.
  • an antibody that binds essentially the same epitope as A12 or B6 antibodies will be referred as an A12-like or B6-like antibody, respectively.
  • the antibodies of the present invention are produced by any technique known in the art, such as, without limitation, any chemical, biological, genetic or enzymatic technique, either alone or in combination.
  • any technique known in the art such as, without limitation, any chemical, biological, genetic or enzymatic technique, either alone or in combination.
  • one skilled in the art can readily produce said antibodies, by standard techniques for production of polypeptides. For instance, they can be synthesized using well-known solid phase method, preferably using a commercially available peptide synthesis apparatus (such as that made by Applied Biosystems, Foster City, California) and following the manufacturer's instructions.
  • antibodies of the present invention can be synthesized by recombinant DNA techniques well-known in the art.
  • a further object of the invention relates to a nucleic acid molecule encoding an antibody according to the invention. More particularly the nucleic acid molecule encodes a heavy chain or a light chain of an antibody of the present invention. More particularly the nucleic acid molecule comprises a nucleic acid sequence having 70% of identity with SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 19. SEQ ID NO: 16 Heavy chain: DNA sequence of A12
  • encoding refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as, for example, a gene, a cDNA, or an m NA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g. , rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • a gene, cDNA, or RNA encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.
  • nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.
  • nucleotide sequence that encodes a protein or a RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).
  • said nucleic acid is a DNA or RNA molecule, which may be included in any suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or a viral vector.
  • a vector such as a plasmid, cosmid, episome, artificial chromosome, phage or a viral vector.
  • vector cloning vector
  • expression vector mean the vehicle by which a DNA or RNA sequence (e.g., a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g., transcription and translation) of the introduced sequence.
  • a further object of the invention relates to a vector comprising a nucleic acid of the invention.
  • Such vectors may comprise regulatory elements, such as a promoter, enhancer, terminator and the like, to cause or direct expression of said antibody upon administration to a subject.
  • promoters and enhancers used in the expression vector for animal cell include early promoter and enhancer of SV40, LTR promoter and enhancer of Moloney mouse leukemia virus, promoter and enhancer of immunoglobulin H chain and the like.
  • Any expression vector for animal cell can be used, so long as a gene encoding the human antibody C region can be inserted and expressed.
  • suitable vectors include pAGE107, pAGE103, pHSG274, pKCR, pSGl beta d2-4 and the like.
  • plasmids include replicating plasmids comprising an origin of replication, or integrative plasmids, such as for instance pUC, pcDNA, pBR, and the like.
  • viral vector include adenoviral, retroviral, herpes virus and AAV vectors.
  • recombinant viruses may be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses.
  • virus packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc.
  • promoter/regulatory sequence refers to a nucleic acid sequence (such as, for example, a DNA sequence) recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence, thereby allowing the expression of a gene product operably linked to the promoter/regulatory sequence.
  • this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product.
  • the promoter/regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner.
  • operably linked refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter.
  • a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • Operably linked DNA sequences can be contiguous with each other and, e.g., where necessary to join two protein coding regions, are in the same reading frame.
  • a further object of the present invention relates to a host cell which has been trans fected, infected or transformed by a nucleic acid and/or a vector according to the invention.
  • transformation means the introduction of a "foreign” (i.e. , extrinsic or extracellular) gene, DNA or RNA sequence to a host cell, so that the host cell will express the introduced gene or sequence to produce a desired substance, typically a protein or enzyme coded by the introduced gene or sequence.
  • a host cell that receives and expresses introduced DNA or RNA bas been "transformed".
  • the nucleic acids of the invention may be used to produce an antibody of the present invention in a suitable expression system.
  • expression system means a host cell and compatible vector under suitable conditions, e.g., for the expression of a protein coded for by foreign DNA carried by the vector and introduced to the host cell.
  • Common expression systems include E. coli host cells and plasmid vectors, insect host cells and Baculovirus vectors, and mammalian host cells and vectors.
  • Other examples of host cells include, without limitation, prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, etc.).
  • E.coli Escherreocoli
  • Kluyveromyces or Saccharomyces yeasts mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.) as well as primary or established mammalian cell cultures (e.g., produced from lymphoblasts, fibroblasts, embryonic cells, epithelial cells, nervous cells, adipocytes, etc.).
  • mammalian cell lines e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.
  • primary or established mammalian cell cultures e.g., produced from lymphoblasts, fibroblasts, embryonic cells, epithelial cells, nervous cells, adipocytes, etc.
  • Examples also include mouse SP2/0-Agl4 cell (ATCC CRL1581), mouse P3X63-Ag8.653 cell (ATCC CRL1580), CHO cell in which a dihydro folate reductase gene (hereinafter referred to as "DHFR gene") is defective (Urlaub G et al; 1980), rat YB2/3HL.P2.G1 1.16Ag.20 cell (ATCC CRL1662, hereinafter referred to as "YB2/0 cell”), and the like.
  • DHFR gene dihydro folate reductase gene
  • the present invention also relates to a method of producing a recombinant host cell expressing an antibody according to the invention, said method comprising the steps of: (i) introducing in vitro or ex vivo a recombinant nucleic acid or a vector as described above into a competent host cell, (ii) culturing in vitro or ex vivo the recombinant host cell obtained and (iii), optionally, selecting the cells which express and/or secrete said antibody.
  • recombinant host cells can be used for the production of antibodies of the present invention.
  • vectors include all those known in the art, including, without limitation, cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.
  • cosmids e.g., naked or contained in liposomes
  • viruses e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses
  • Antibodies of the present invention are suitably separated from the culture medium by conventional immunoglobulin purification procedures such as, for example, protein A- Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • the present invention further relates to a composition
  • a composition comprising, consisting of or consisting essentially of an antibody of the present invention, preferably a B6-like or A12-like antibody.
  • composition consisting essentially of, with reference to a composition, means that the at least one antibody of the invention as described hereinabove is the only one therapeutic agent or agent with a biologic activity within said composition.
  • the composition of the invention is a pharmaceutical composition and further comprises a pharmaceutically acceptable carrier.
  • the present invention further relates to a medicament comprising, consisting of or consisting essentially of an antibody of the present invention, preferably a B6-like or A12-like antibody.
  • Engineered antibodies of the present invention include those in which modifications have been made to framework residues within VH and/or VL, e.g. , to improve the properties of the antibody. Typically such framework modifications are made to decrease the immunogenicity of the antibody. For example, one approach is to "backmutate" one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequences to the germline sequences from which the antibody is derived.
  • the somatic mutations can be "backmutated” to the germline sequence by, for example, site- directed mutagenesis or PCR-mediated mutagenesis.
  • Such "backmutated” antibodies are also intended to be encompassed by the invention.
  • Another type of framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T cell-epitopes to thereby reduce the potential immunogenicity of the antibody. This approach is also referred to as "deimmunization" and is described in further detail in U.S. Patent Publication No. 20030153043 by Carr et al.
  • the antibody (preferably the monoclonal antibody) of the present invention comprises a Fc region that mediates antibody-dependent cell-mediated cytotoxicity.
  • antibody-dependent cell-mediated cytotoxicity or 'ADCC refer to a cell-mediated reaction in which non-specific cytotoxic cells (e.g. , Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cell. While not wishing to be limited to any particular mechanism of action, these cytotoxic cells that mediate ADCC generally express Fc receptors (FcRs).
  • FcRs Fc receptors
  • Fc region includes the polypeptides comprising the constant region of an antibody excluding the first constant region immunoglobulin domain.
  • Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains.
  • IgA and IgM Fc may include the J chain.
  • Fc comprises immunoglobulin domains Cgamma2 and Cgamma3 (Cy2 and Cy3) and the hinge between Cgammal (Cyl) and Cgamma2 (Cy2).
  • the human IgG heavy chain Fc region is usually defined to comprise residues C226 or P230 to its carboxyl-terminus, wherein the numbering is according to the EU index as in Kabat et al. ( 1991 , NIH Publication 91-3242, National Technical Information Service, Springfield, Va.).
  • the "EU index as set forth in Kabat” refers to the residue numbering of the human IgGl EU antibody as described in Kabat et al. supra.
  • Fc may refer to this region in isolation, or this region in the context of an antibody, antibody fragment, or Fc fusion protein.
  • An Fc variant protein may be an antibody, Fc fusion, or any protein or protein domain that comprises an Fc region.
  • proteins comprising variant Fc regions, which are non-naturally occurring variants of an Fc region.
  • the amino acid sequence of a non-naturally occurring Fc region (also referred to herein as a "variant Fc region”) comprises a substitution, insertion and/or deletion of at least one amino acid residue compared to the wild type amino acid sequence. Any new amino acid residue appearing in the sequence of a variant Fc region as a result of an insertion or substitution may be referred to as a non-naturally occurring amino acid residue.
  • Polymorphisms have been observed at a number of Fc positions, including but not limited to Kabat 270, 272, 312, 315, 356, and 358, and thus slight differences between the presented sequence and sequences in the prior art may exist.
  • Fc receptor or "FcR” are used to describe a receptor that binds to the Fc region of an antibody.
  • the primary cells for mediating ADCC NK cells, express FcyRIII, whereas monocytes express FcyRI, FcyRII, FcyRIII and/or FcyRIV.
  • FcR expression on hematopoietic cells is summarized in Ravetch and Kinet, Annu. Rev. Immunol., 9:457-92 (1991).
  • an in vitro ADCC assay such as that described in U.S. Pat. No. 5,500,362 or 5,821,337 may be performed.
  • effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
  • PBMC peripheral blood mononuclear cells
  • NK Natural Killer
  • ADCC activity of the molecules of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., Proc. Natl. Acad. Sci. (USA), 95:652-656 (1998).
  • effector cells are leukocytes which express one or more FcRs and perform effector functions. The cells express at least FcyRI, FCyRII, FcyRIII and/or FcyRIV and carry out ADCC effector function.
  • human leukocytes which mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils.
  • the antibody (preferably the monoclonal antibody) of the present invention is a full-length antibody.
  • the full-length antibody is an IgGl antibody.
  • the full-length antibody is an IgG3 antibody.
  • the antibody (preferably the monoclonal antibody) of the present invention comprises a variant Fc region that has an increased affinity for FcyRIA, FcyRIIA, FcyRIIB, FcyRIIIA, FcyRIIIB, and FcyRIV.
  • the antibody (preferably the monoclonal antibody) of the present invention comprises a variant Fc region comprising at least one amino acid substitution, insertion or deletion wherein said at least one amino acid residue substitution, insertion or deletion results in an increased affinity for FcyRIA, FcyRIIA, FcyRIIB, FcyRIIIA, FcyRIIIB, and FcyRIV.
  • the antibody (preferably the monoclonal antibody) of the present invention comprises a variant Fc region comprising at least one amino acid substitution, insertion or deletion wherein said at least one amino acid residue is selected from the group consisting of: residue 239, 330, and 332, wherein amino acid residues are numbered following the EU index.
  • the antibody (preferably the monoclonal antibody) of the present invention comprises a variant Fc region comprising at least one amino acid substitution wherein said at least one amino acid substitution is selected from the group consisting of: S239D, A330L, A330Y, and 1332E, wherein amino acid residues are numbered following the EU index.
  • the glycosylation of the antibody of the present invention is modified.
  • an aglycoslated antibody can be made (i.e., the antibody lacks glycosylation).
  • Glycosylation can be altered to, for example, increase the affinity of the antibody for the antigen or alter the ADCC activity of the antibody.
  • Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence.
  • one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site.
  • Such aglycosylation may increase the affinity of the antibody for antigen.
  • an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated or non-fucosylated antibody having reduced amounts of or no fucosyl residues or an antibody having increased bisecting GlcNac structures.
  • Such altered fucosylation patterns have been demonstrated to increase the ADCC ability of antibodies.
  • Such carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies of the present invention to thereby produce an antibody with altered glycosylation.
  • EP 1176195 by Hang et al. describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation or are devoid of fucosyl residues. Therefore, in some embodiments, the human antibody (preferably the monoclonal antibody) of the present invention may be produced by recombinant expression in a cell line which exhibit hypofucosylation or non- fucosylation pattern, for example, a mammalian cell line with deficient expression of the FUT8 gene encoding fucosyltransferase.
  • PCT Publication WO 03/035835 by Presta describes a variant CHO cell line, Lecl3 cells, with reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell (see also Shields, R.L. et al, 2002 J. Biol. Chem. 277:26733-26740).
  • glycoprotein-modifying glycosyl transferases e.g., beta(l,4)-N acetylglucosaminyltransferase III (GnTIII)
  • GnTIII glycoprotein-modifying glycosyl transferases
  • Eureka Therapeutics further describes genetically engineered CHO mammalian cells capable of producing antibodies with altered mammalian glycosylation pattern devoid of fucosyl residues (http ://www. eurekainc.com/ a&boutus/ companyoverview.html) .
  • the human antibody (preferably the monoclonal antibody) of the present invention can be produced in yeasts or filamentous fungi engineered for mammalian-like glycosylation pattern and capable of producing antibodies lacking fucose as glycosylation pattern (see for example EP1297172B1).
  • the antibody (preferably the monoclonal antibody) of the present invention comprises a Fc region that mediates complement dependant cytotoxicity.
  • complement dependent cytotoxicity or “CDC” refers to the ability of a molecule to initiate complement activation and lyse a target in the presence of complement.
  • the complement activation pathway is initiated by the binding of the first component of the complement system (C lq) to a molecule (e.g., an antibody) complexed with a cognate antigen.
  • a CDC assay e.g., as described in Gazzano-Santaro et al., J. Immunol. Methods, 202: 163 (1996), may be performed.
  • the antibody (preferably the monoclonal antibody) of the present invention comprises a Fc region that mediates antibody-dependent phagocytosis.
  • antibody-dependent phagocytosis or “opsonisation” refers to the cell- mediated reaction wherein nonspecific cytotoxic cells that express FcyRs recognize bound antibody on a target cell and subsequently cause phagocytosis of the target cell.
  • the antibody (preferably the monoclonal antibody) of the present invention comprises a Fc region inducing ADCC or CDC or antibody-dependent phagocytosis. Consequently, administration of such antibody to a subject may lead to the depletion of cells expressing CD160-TM (e.g., leads to a 10%, 20%, 50%>, 60%> or greater elimination or decrease in number of CD 160-TM + NK cells), such as, for example CD160-TM expressing tumor cells.
  • a further object of the present invention thus relates to a method of depleting a population of cells which express the CD160-TM isoform in a subject in need thereof comprising delivering to the subject a therapeutically effective amount of the antibody (preferably the monoclonal antibody) of the present invention.
  • the antibody (preferably the monoclonal antibody) of the present invention comprises a Fc region inducing ADCC or CDC or antibody-dependent phagocytosis.
  • a further object of the present invention relates to a method of depleting a population of malignant NK cells which express the CD160-TM isoform in a subject in need thereof comprising delivering to the subject a therapeutically effective amount of the antibody (preferably the monoclonal antibody) of the present invention.
  • the antibody (preferably the monoclonal antibody) of the present invention comprises a Fc region inducing ADCC or CDC or antibody-dependent phagocytosis.
  • a further object of the present invention relates to a method of depleting a population of cells which express the epitope recognized by the A12 or B6 antibody in in a subject in need thereof comprising delivering to the subject a therapeutically effective amount of the antibody (preferably the monoclonal antibody) of the present invention.
  • the antibody (preferably the monoclonal antibody) of the present invention comprises a Fc region inducing ADCC or CDC or antibody-dependent phagocytosis.
  • the term “deplete” with respect to a population of cells refers to a measurable decrease in the number of said cells in the subject.
  • the reduction can be at least about 10%, e.g. , at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more.
  • the term refers to a decrease in the number of the cells in a subject or in a sample to an amount below detectable limits.
  • the antibody (preferably the monoclonal antibody) of the present invention mediates antibody dependent cellular cytotoxicity, complement dependent cytotoxicity and antibody-dependent phagocytosis.
  • a further object of the present invention relates to a method of treating a cancer wherein cancer cells express CD160-TM.
  • cancers wherein cancer cells express CD160-TM include, but are not limited to, a NK leukemia or a NK lymphoma, such as for example, extranodal and non-extranodal NK/T lymphomas; NK cell derived malignancies; and acute NK leukemia.
  • the present invention thus further relates to an antibody, a composition, a pharmaceutical composition or a medicament of the present invention for use in treating a cancer wherein cancer cells express CD160-TM.
  • the antibody (preferably the monoclonal antibody) of the present invention does not comprise a Fc region that mediates antibody-dependent cell-mediated cytotoxicity and thus does not comprise an Fc portion that induces antibody dependent cellular cytotoxicity (ADCC).
  • the antibody (preferably the monoclonal antibody) of the present invention does not comprise an Fc region that induces CDC or antibody- dependent phagocytosis.
  • the antibody (preferably the monoclonal antibody) of the present invention does not lead, directly or indirectly, to the depletion of NK cells expressing CD160-TM polypeptides (e.g., do not lead to a 10%, 20%, 50%>, 60%> or greater elimination or decrease in number of CD 160-TM + NK cells).
  • the antibody (preferably the monoclonal antibody) of the present invention does not comprise an Fc domain capable of substantially binding to a FcyRIIIA (CD 16) polypeptide.
  • the antibody (preferably the monoclonal antibody) of the present invention lacks an Fc domain (e.g., lacks a CH2 and/or CH3 domain) or comprises an Fc domain of IgG2 or IgG4 isotype. In some embodiments, the antibody (preferably the monoclonal antibody) of the present invention comprises an Fc domain (e.g. of IgGl) with an altered glycosylation profile, resulting in the absence of ADCC activity of the antibody.
  • the antibody (preferably the monoclonal antibody) of the present invention consists of or comprises a Fab, Fab', Fab'-SH, F(ab') 2 , Fv, a diabody, single-chain antibody fragment, or a multispecific antibody comprising multiple different antibody fragments.
  • the antibody (preferably the monoclonal antibody) of the present invention is not linked to a toxic moiety.
  • one or more amino acids selected from amino acid residues can be replaced with a different amino acid residue such that the antibody has altered C2q binding and/or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Patent Nos. 6, 194,551 by ldusogie et al.
  • a further object of the present invention relates to a method of enhancing NK cell activities or NK cell effector functions, in particular NK cell killing activities in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an antibody of the present invention, provided that the antibody does not mediate antibody dependent cellular cytotoxicity, complement dependent cytotoxicity or antibody-dependent phagocytosis.
  • NK cells refers to a sub-population of lymphocytes that is involved in innate or non-conventional immunity. NK cells can be identified by virtue of certain characteristics and biological properties, such as the expression of specific surface antigens including CD56 and/or CD 16 for human NK cells, the absence of the alpha/beta or gamma/delta TCR complex on the cell surface, the ability to bind to and kill cells that fail to express "self MHC/HLA antigens by the activation of specific cytolytic machinery, the ability to kill tumor cells or other diseased cells that express a ligand for NK activating receptors, and the ability to release protein molecules called cytokines that stimulate or inhibit the immune response ("NK cell activities").
  • specific surface antigens including CD56 and/or CD 16 for human NK cells
  • the absence of the alpha/beta or gamma/delta TCR complex on the cell surface the ability to bind to and kill cells that fail to express "self MHC/HLA antigens by the activation
  • NK cells any subpopulation of NK cells will also be encompassed by the termNK cells.
  • active NK cells designate biologically active NK cells, including NK cells having the capacity of lysing target cells or enhancing the immune function of other cells.
  • an "active" NK cell can be able to kill cells that express a ligand for an activating NK receptor and/or fail to express MHC/HLA antigens recognized by a KIR on the NK cell.
  • the ability of the antibody (preferably the monoclonal antibody) of the present invention to enhance NK cell activities, in particular NK cell killing activities, may be determined by any assay well known in the art.
  • said assay is an in vitro assay wherein NK cells are brought into contact with target cells (e.g., target cells that are recognized and/or lysed by NK cells).
  • target cells e.g., target cells that are recognized and/or lysed by NK cells.
  • the antibody can be selected for the ability to increase specific lysis by NK cells by more than about 20%, preferably with at least about 30%, at least about 40%, at least about 50%, or more of the specific lysis obtained at the same effector: target cell ratio with NK cells or NK cell lines that are contacted by the antibody (preferably the monoclonal antibody) of the present invention,.
  • NK cell cytotoxicity is determined by any assay described in the EXAMPLE.
  • NK cell cytotoxicity may be measured by a classical in vitro chromium release test of cytotoxicity.
  • Effector cells are typically fresh PB-NK from healthy donors.
  • the target cells are typically the murine mastocytoma P815 cells or EBV-infected B cell lines.
  • the antibody (preferably the monoclonal antibody) of the present invention is selected if it causes an increase in the reactivity or cytoxicity of NK cells toward target cells (infected cells, tumor cells, pro-inflammatory cells, etc.), increased activation, activation markers (e.g., CD107 expression) and/or IFNgamma production in NK cells, and/or increased the frequency in vivo of such activated, reactive, cytotoxic and/or activated NK cells.
  • the subject suffers from a cancer or an infectious disease.
  • a further object of the present invention relates to a method of treating a cancer or an infectious disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an antibody of the present invention.
  • the antibody of the present invention does not comprise a Fc region mediating ADCC, CDC or antibody-induced phagocytosis.
  • the present invention thus further relates to an antibody, a composition, a pharmaceutical composition or a medicament of the present invention for use in treating a cancer, an infectious disease or an inflammatory and/or auto-immune disease.
  • treatment is an approach for obtaining beneficial or desired results including clinical results.
  • beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g. , preventing or delaying the worsening of the disease), preventing or delaying the spread (e.g.
  • treatment is a reduction of pathological consequence of cancer.
  • the methods of the present invention contemplate any one or more of these aspects of treatment.
  • the terms "treating" or “treatment” refers to both therapeutic treatment and prophylactic or preventative measures; wherein the object is to prevent or slow down (lessen) the targeted disease. Therefore, in one embodiment, those in need of treatment may include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented.
  • cancer has its general meaning in the art and includes, but is not limited to, solid tumors and blood borne tumors
  • the term cancer includes diseases of the skin, tissues, organs, bone, cartilage, blood and vessels.
  • the term “cancer” further encompasses both primary and metastatic cancers. Examples of cancers that may treated by methods and compositions of the invention include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus.
  • the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acid
  • the term "infectious disease” includes any infection caused by viruses, bacteria, protozoa, molds or fungi.
  • the viral infection comprises infection by one or more viruses selected from the group consisting of Arenaviridae, Astroviridae, Birnaviridae, Bromoviridae, Bunyaviridae, Caliciviridae, Closteroviridae, Comoviridae, Cystoviridae, Flaviviridae, Flexiviridae, Hepevirus, Leviviridae, Luteoviridae, Mononegavirales, Mosaic Viruses, Nidovirales, Nodaviridae, Orthomyxoviridae, Picobirnavirus, Picornaviridae, Potyviridae, Reoviridae, Retroviridae, Sequiviridae, Tenuivirus, Togaviridae, Tombusviridae, Totiviridae, Tymoviridae, Hepadnaviridae, vac
  • RNA viruses include, without limitation, Astroviridae, Birnaviridae, Bromoviridae, Caliciviridae, Closteroviridae, Comoviridae, Cystoviridae, Flaviviridae, Flexiviridae, Hepevirus, Leviviridae, Luteoviridae, Mononegavirales, Mosaic Viruses, Nidovirales, Nodaviridae, Orthomyxoviridae, Picobirnavirus, Picornaviridae, Potyviridae, Reoviridae, Retroviridae, Sequiviridae, Tenuivirus, Togaviridae, Tombusviridae, Totiviridae, and Tymoviridae viruses.
  • the viral infection comprises infection by one or more viruses selected from the group consisting of adenovirus, rhinovirus, hepatitis, immunodeficiency virus, polio, measles, Ebola, Coxsackie, Pvhino, West Nile, small pox, encephalitis, yellow fever, Dengue fever, influenza (including human, avian, and swine), lassa, lymphocytic choriomeningitis, junin, machuppo, guanarito, hantavirus, Rift Valley Fever, La Crosse, California encephalitis, Crimean-Congo, Marburg, Japanese Encephalitis, Kyasanur Forest, Venezuelan equine encephalitis, Eastern equine encephalitis, Western equine encephalitis, severe acute respiratory syndrome (SARS), parainfluenza, respiratory syncytial, Punta Toro, Tacaribe, pachindae viruses, adeno
  • viruses selected
  • Bacterial infections that can be treated according to this invention include, but are not limited to, infections caused by the following: Staphylococcus; Streptococcus, including S. pyogenes; Enterococcl; Bacillus, including Bacillus anthracis, and Lactobacillus; Listeria; Corynebacterium diphtheriae; Gardnerella including G.
  • vaginalis Nocardia; Streptomyces; Thermoactinomyces vulgaris; Treponema; Camplyobacter, Pseudomonas including aeruginosa; Legionella; Neisseria including N. gonorrhoeae and Nmeningitides; Flavobacterium including F. meningosepticum and F. odoraturn; Brucella; Bordetella including B. pertussis and B. bronchiseptica; Escherichia including E. coli, Klebsiella; Enterobacter, Serratia including S. marcescens and S. Uquefaciens; Edwardsiella; Proteus including P. mirabilis and P.
  • Protozoa infections that may be treated according to this invention include, but are not limited to, infections caused by leishmania, kokzidioa, and trypanosoma.
  • NCID National Center for Infectious Disease
  • CDC Center for Disease Control
  • All of said diseases are candidates for treatment using the compositions according to the invention.
  • inflammatory diseases include, but are not limited to, arthritis, rheumatoid arthritis, ankylosing spondylitis, osteoarthritis, psoriatic arthritis, juvenile idiopathic arthritis, juvenile rheumatoid arthritis, arthritis uratica, gout, chronic polyarthritis, periarthritis humeroscapularis, cervical arthritis, lumbosacral arthritis, enteropathic arthritis and ankylosing spondylitis, asthma, dermatitis, psoriasis, scleroderma, polymyositis, dermatomyositis, juvenila dermatomyositis, primary biliary cirrhosis, fibrosis, cystic fibrosis, pulmonary fibrosis, cirrhosis, endomyocardial fibrosis, dediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, nephrogenic fibros,
  • autoimmune diseases include, but are not limited to, lupus (e.g., lupus erythematosus, lupus nephritis), Hashimoto's thyroiditis, Wegener's disease; primary myxedema, Graves' disease, pernicious anemia, autoimmune atrophic gastritis, Addison's disease, diabetes (e.g.
  • insulin dependent diabetes mellitus type I diabetes mellitus, type II diabetes mellitus
  • good pasture's syndrome myasthenia gravis, pemphigus
  • intestinal inflammatory conditions such as Crohn's disease and ulcerative colitis
  • sympathetic ophthalmia autoimmune uveitis, multiple sclerosis, autoimmune hemolytic anemia, idiopathic thrombocytopenia, primary biliary cirrhosis, chronic action hepatitis, ulcerative colitis, Sjogren's syndrome, arthritis conditions such as rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis and juvenile idiopathic arthritis
  • asthma transplant rejection (host versus graft disease); graft versus host disease and mixed connective tissue disease.
  • the present invention also provides for therapeutic applications where an antibody of the present invention is used in combination with at least one further therapeutic agent, e.g., for treating cancer.
  • Such administration may be simultaneous, separate or sequential.
  • the agents may be administered as one composition or as separate compositions, as appropriate.
  • the further therapeutic agent is typically relevant for the disorder to be treated.
  • Exemplary therapeutic agents include other anti-cancer antibodies, cytotoxic agents, chemotherapeutic agents, anti-angiogenic agents, anti-cancer immunogens, cell cycle control/apoptosis regulating agents, hormonal regulating agents, and other agents described below.
  • the second agent is a natural ligand of an NK cell activating or an antibody that binds and activates an NK cell activating receptor other than CD160-TM.
  • the agent is an agent that increases the presence of a natural ligand of an NK cell activating receptor on the surface of a target cell (e.g., infected cells, or tumor cells).
  • the antibody of the present invention does not comprise a Fc region mediating ADCC, CDC or antibody-induced phagocytosis and is used in combination with a second agent that (i) is a natural ligand of an NK cell activating or an antibody that binds and activates anNK cell activating receptor other than CD160-TM and/or (ii) increases the presence of a natural ligand of an NK cell activating receptor on the surface of a target cell.
  • NK cell activating receptors include, for example, NKG2D or activating KIR receptors (KIR2DS receptors, KIR2DS2, KIR2DS4).
  • activating NK receptor refers to any molecule on the surface of NK cells that, when stimulated, causes a measurable increase in any property or activity known in the art as associated with NK activity, such as cytokine (for example IFN- ⁇ and TNF-a) production, increases in intracellular free calcium levels, the ability to target cells in a redirected killing assay as described, e.g., elsewhere in the present specification, or the ability to stimulate NK cell proliferation.
  • cytokine for example IFN- ⁇ and TNF-a
  • the term “activating NK receptor” includes but is not limited to activating forms or KIR proteins (for example KIR2DS proteins), NKG2D, IL-2R, IL-12R, IL-15R, IL-18R and IL-21R.
  • CD 137 examples include, e.g., IL-2, IL-15, IL-21 polypeptides.
  • the second antibody is specific for CD 137.
  • CD 137 has its general meaning in the art and may also be referred to as Ly63, ILA or 4- IBB.
  • CD 137 is a member of the tumor necrosis factor (TNF) receptor family. Members of this receptor family and their structurally related ligands are important regulators of a wide variety of physiologic processes and play an important role in the regulation of immune responses.
  • TNF tumor necrosis factor
  • CD 137 is expressed by activated NK cells, T and B lymphocytes and monocytes/macrophages.
  • the gene encodes a 255 -amino acid protein with 3 cysteine-rich motifs in the extracellular domain (characteristic of this receptor family), a transmembrane region, and a short N- terminal cytoplasmic portion containing potential phosphorylation sites. Expression in primary cells is strictly activation dependent.
  • the ligand for the receptor is TNFSF9. Human CD 137 is reported to bind only to its ligand. Agonists include the native ligand (TNFSF9), aptamers (see McNamara et al. (2008) J. Clin. Invest. 1 18: 376-386), and antibodies.
  • antibody of the present invention is used in combination with a second antibody which induces, via ADCC, the death of a cell expressing an antigen to which the second antibody binds.
  • the antibody of the present invention does not comprise a Fc region mediating ADCC, CDC or antibody-induced phagocytosis and is used in combination with a second agent which induces, via ADCC, the death of a cell expressing an antigen to which the second antibody binds.
  • the antibody of the invention is conjugated to a second antibody which induces, via ADCC, the death of a cell expressing an antigen to which the second antibody binds.
  • NK cells have an important role in inducing ADCC and increased reactivity of NK cells can be directed to target cells through use of such a second agent.
  • the second agent is an antibody specific for a cell surface antigens, e.g., membrane antigens.
  • the second antibody is specific for a tumor antigen as described herein (e.g., molecules specifically expressed by tumor cells), such as CD20, CD52, ErbB2 (or HER2/Neu), CD33, CD22, CD25, MUC-1 , CEA, KDR, aV 3, etc., particularly lymphoma antigens (e.g., CD20).
  • the present invention also provides methods to enhance the anti-tumor effect of monoclonal antibodies directed against tumor antigen(s).
  • ADCC function is specifically augmented, which in turn enhances target cell killing, by sequential administration of an antibody directed against one or more tumor antigens, and an antibody of the present invention.
  • a further object relates to a method of enhancing NK cell antibody- dependent cellular cytotoxicity (ADCC) of an antibody in a subject in need thereof comprising administering to the subject the antibody, and administering to the subject an antibody of the present invention, wherein preferably the antibody of the present invention does not comprise a Fc region mediating ADCC, CDC or antibody-induced phagocytosis.
  • ADCC NK cell antibody- dependent cellular cytotoxicity
  • a further object of the present invention relates to a method of treating cancer in a subject in need thereof comprising administering to the subject a first antibody selective for a cancer cell antigen, and administering to the subject an antibody of the present invention, wherein preferably the antibody of the present invention does not comprise a Fc region mediating ADCC, CDC or antibody-induced phagocytosis.
  • Antibodies of interest for the methods of the invention act through ADCC, and are typically selective for tumor cells, although one of skill in the art will recognize that some clinically useful antibodies do act on non-tumor cells, e.g. , CD20.
  • CD20 There are a number of antigens and corresponding monoclonal antibodies for the treatment of B cell malignancies.
  • One popular target antigen is CD20, which is found on B cell malignancies.
  • Rituximab is a chimeric unconjugated monoclonal antibody directed at the CD20 antigen.
  • CD20 has an important functional role in B cell activation, proliferation, and differentiation.
  • the CD52 antigen is targeted by the monoclonal antibody alemtuzumab, which is indicated for treatment of chronic lymphocytic leukemia.
  • CD22 is targeted by a number of antibodies, and has recently demonstrated efficacy combined with toxin in chemotherapy- resistant hairy cell leukemia.
  • Monoclonal antibodies targeting CD20 also include tositumomab and ibritumomab.
  • Monoclonal antibodies useful in the methods of the invention, which have been used in solid tumors include without limitation edrecolomab and trastuzumab (herceptin).
  • Edrecolomab targets the 17-1 A antigen seen in colon and rectal cancer, and has been approved for use in Europe for these indications.
  • Trastuzumab targets the HER- 2/neu antigen. This antigen is seen on 25% to 35% of breast cancers. Trastuzumab is thought to work in a variety of ways: downregulation of HER-2 receptor expression, inhibition of proliferation of human tumor cells that overexpress HER-2 protein, enhancing immune recruitment and ADCC against tumor cells that overexpress HER-2 protein, and downregulation of angiogenesis factors.
  • Alemtuzumab (Campath) is used in the treatment of chronic lymphocytic leukemia; colon cancer and lung cancer; Gemtuzumab (Mylotarg) finds use in the treatment of acute myelogenous leukemia; Ibritumomab (Zevalin) finds use in the treatment of non-Hodgkin's lymphoma; Panitumumab (Vectibix) finds use in the treatment of colon cancer. Cetuximab (Erbitux) is also of interest for use in the methods of the invention.
  • the antibody binds to the EGF receptor (EGFR), and has been used in the treatment of solid tumors including colon cancer and squamous cell carcinoma of the head and neck (SCCHN).
  • EGFR EGF receptor
  • the antibody (preferably the monoclonal antibody) of the present invention is used in combination with at least one immune checkpoint inhibitor (ICI).
  • ICI immune checkpoint inhibitor
  • Various tumors are able to express molecular factors protecting them from being attacked by the immune system, and are thus capable of successfully escaping the immune system supervision control. This "tumor immune escape” is mainly due to the antagonistic blocking of receptors and binding sites targeted by immune cell ligands.
  • Immune checkpoint inhibitors are molecules especially targeting this kind of inhibitory mechanisms developed by tumorous cells.
  • ICIs include, but are not limited to, inhibitors of CTLA-4 (such as, for example, ipilumab and tremelimumab), inhibitors of PD-1 (such as, for example, pembrolizumab, pidilizumab, nivolumab and AMP-224) inhibitors of PD-L1 (such as, for example, atezolizumab, avelumab, durvalumab and BMS-936559), inhibitors of LAG3 (such as, for example, IMP321) and inhibitors of B7-H3 (such as, for example, MGA271).
  • the antibody of the present invention does not comprise a Fc region mediating ADCC, CDC or antibody-induced phagocytosis and is used in combination with at least one immune checkpoint inhibitor (ICI).
  • the antibody (preferably the monoclonal antibody) of the present invention inhibits the binding of CD160-TM to one of its ligands, such as, for example, to MHC class I molecules.
  • ligand refers to a member of a pair ligand/receptor, and binds to the other member of the pair. Inhibiting the binding of CD160-TM to one of its ligands may thus inhibit the functionality of NK cells expressing CD160-TM.
  • Such inhibition may be useful for treating Paroxysmal Nocturnal Hemoglobinuria or an inflammatory and/or autoimmune disease.
  • inflammatory and/or autoimmune disease are listed hereinabove.
  • a further object of the present invention thus relates to a method of treating Paroxysmal Nocturnal Hemoglobinuria in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the antibody (preferably the monoclonal antibody) of the present invention.
  • the term "Paroxysmal Nocturnal Hemoglobinuria" or "PNH” has its general meaning in the art and refers to an acquired clonal hematopoietic stem cell disorder characterized by corpuscular hemolytic anemia, bone marrow failure and frequent thrombotic events.
  • the subject is not mutated for the PIGA gene (phosphatidylinositol glycan anchor biosynthesis class A, Gene ID: 5277).
  • the present invention thus further relates to an antibody, a composition, a pharmaceutical composition or a medicament for use in the treatment of PNH.
  • the antibody (preferably the monoclonal antibody) of the present invention does not comprise a Fc region mediating ADCC, CDC or antibody-induced phagocytosis.
  • the antibody (preferably the monoclonal antibody) of the present invention consists of or comprises a Fab, Fab', Fab'-SH, F(ab') 2 , Fv, a diabody, single-chain antibody fragment, or a multispecific antibody comprising multiple different antibody fragments.
  • the antibody (preferably the monoclonal antibody) of the present invention is a Fab.
  • the present invention further relates to a fusion protein comprising an antibody (preferably a monoclonal antibody) of the present invention.
  • the antibody (preferably the monoclonal antibody) of the present invention is conjugated to a therapeutic moiety, i.e., a drug.
  • the therapeutic moiety can be, e.g., a cytotoxin, a chemotherapeutic agent, a cytokine, an immunosuppressant, an immune stimulator, a lytic peptide, or a radioisotope.
  • conjugates are referred to herein as an "antibody-drug conjugates" or "ADCs".
  • the antibody of the present invention does not comprise a Fc region mediating ADCC, CDC or antibody-induced phagocytosis and is conjugated to a therapeutic moiety.
  • the antibody (preferably the monoclonal antibody) of the present invention is conjugated to a cytotoxic moiety.
  • the antibody of the present invention does not comprise a Fc region mediating ADCC, CDC or antibody-induced phagocytosis and is conjugated to a cytotoxic moiety.
  • the cytotoxic moiety may, for example, be selected from the group consisting of taxol; cytochalasin B; gramicidin D; ethidium bromide; emetine; mitomycin; etoposide; tenoposide; vincristine; vinblastine; colchicin; doxorubicin; daunorubicin; dihydroxy anthracin dione; a tubulin- inhibitor such as maytansine or an analog or derivative thereof; an antimitotic agent such as monomethyl auristatin E or F or an analog or derivative thereof; dolastatin 10 or 15 or an analogue thereof; irinotecan or an analogue thereof; mit
  • the antibody (preferably the monoclonal antibody) of the present invention is conjugated to an auristatin or a peptide analog, derivative or prodrug thereof.
  • the antibody of the present invention does not comprise a Fc region mediating ADCC, CDC or antibody-induced phagocytosis and is conjugated to an auristatin or a peptide analog, derivative or prodrug thereof.
  • Auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis and nuclear and cellular division (Woyke et al (2001) Antimicrob. Agents and Chemother.
  • auristatin E can be reacted with para-acetyl benzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively.
  • Other typical auristatin derivatives include AFP, MMAF (monomethyl auristatin F), and MMAE (monomethyl auristatin E).
  • Suitable auristatins and auristatin analogs, derivatives and prodrugs, as well as suitable linkers for conjugation of auristatins to Abs are described in, e.g. , U.S. Patent Nos. 5,635,483, 5,780,588 and 6,214,345 and in International patent application publications WO02088172, WO2004010957, WO200508171 1 , WO2005084390, WO2006132670, WO03026577, WO200700860, WO20701 1968 and WO205082023.
  • the antibody (preferably the monoclonal antibody) of the present invention is conjugated to pyrrolo[2,l-c][l,4]- benzodiazepine (PDB) or an analog, derivative or prodrug thereof.
  • the antibody of the present invention does not comprise a Fc region mediating ADCC, CDC or antibody-induced phagocytosis and is conjugated to a PDB or an analog, derivative or prodrug thereof.
  • Suitable PDBs and PDB derivatives, and related technologies are described in, e.g. , Hartley J. A. et al, Cancer Res 2010; 70(17) : 6849- 6858; Antonow D.
  • the antibody (preferably the monoclonal antibody) of the present invention is conjugated to a cytotoxic moiety selected from the group consisting of an anthracycline, maytansine, calicheamicin, duocarmycin, rachelmycin (CC-1065), dolastatin 10, dolastatin 15, irinotecan, monomethyl auristatin E, monomethyl auristatin F, a PDB, or an analog, derivative, or prodrug of any thereof.
  • a cytotoxic moiety selected from the group consisting of an anthracycline, maytansine, calicheamicin, duocarmycin, rachelmycin (CC-1065), dolastatin 10, dolastatin 15, irinotecan, monomethyl auristatin E, monomethyl auristatin F, a PDB, or an analog, derivative, or prodrug of any thereof.
  • the antibody of the present invention does not comprise a Fc region mediating ADCC, CDC or antibody-induced phagocytosis and is conjugated to a cytotoxic moiety selected from the group consisting of an anthracycline, maytansine, calicheamicin, duocarmycin, rachelmycin (CC-1065), dolastatin 10, dolastatin 15, irinotecan, monomethyl auristatin E, monomethyl auristatin F, a PDB, or an analog, derivative, or prodrug of any thereof.
  • a cytotoxic moiety selected from the group consisting of an anthracycline, maytansine, calicheamicin, duocarmycin, rachelmycin (CC-1065), dolastatin 10, dolastatin 15, irinotecan, monomethyl auristatin E, monomethyl auristatin F, a PDB, or an analog, derivative, or prodrug of any thereof.
  • the antibody (preferably the monoclonal antibody) of the present invention is conjugated to an anthracycline or an analog, derivative or prodrug thereof. In some embodiments, the antibody is conjugated to maytansine or an analog, derivative or prodrug thereof. In some embodiments, the antibody is conjugated to calicheamicin or an analog, derivative or prodrug thereof. In some embodiments, the antibody is conjugated to duocarmycin or an analog, derivative or prodrug thereof. In some embodiments, the antibody is conjugated to rachelmycin (CC-1065) or an analog, derivative or prodrug thereof. In some embodiments, the antibody is conjugated to dolastatin 10 or an analog, derivative or prodrug thereof.
  • the antibody is conjugated to dolastatin 15 or an analog, derivative or prodrug thereof. In some embodiments, the antibody is conjugated to monomethyl auristatin E or an analog, derivative or prodrug thereof. In some embodiments, the antibody is conjugated to monomethyl auristatin F or an analog, derivative or prodrug thereof. In some embodiments, the antibody is conjugated to pyrrolo[2,l-c][l,4]-benzodiazepine or an analog, derivative or prodrug thereof. In some embodiments, the antibody is conjugated to irinotecan or an analog, derivative or prodrug thereof.
  • nucleic acid molecule is covalently attached to lysines or cysteines on the antibody, through N- hydroxysuccinimide ester or maleimide functionality respectively.
  • TDCs cysteine-based site-specific conjugation
  • ADCs cysteine-based site-specific conjugation
  • Conjugation to unnatural amino acids that have been incorporated into the antibody is also being explored for ADCs; however, the generality of this approach is yet to be established (Axup et al., 2012).
  • Fc-containing polypeptide engineered with an acyl donor glutamine-containing tag e.g., Gin-containing peptide tags or Q- tags
  • an endogenous glutamine that are made reactive by polypeptide engineering (e.g. , via amino acid deletion, insertion, substitution, or mutation on the polypeptide).
  • a transglutaminase can covalently crosslink with an amine donor agent (e.g., a small molecule comprising or attached to a reactive amine) to form a stable and homogenous population of an engineered Fc-containing polypeptide conjugate with the amine donor agent being site- specifically conjugated to the Fc-containing polypeptide through the acyl donor glutamine- containing tag or the accessible/exposed/reactive endogenous glutamine (WO 2012059882).
  • an amine donor agent e.g., a small molecule comprising or attached to a reactive amine
  • the term "therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result.
  • the terms “therapeutically effective amount” may mean a level or amount of antibodies that is aimed at, without causing significant negative or adverse side effects to the target, (1) delaying or preventing the onset of the targeted disease; (2) slowing down or stopping the progression, aggravation, or deterioration of one or more symptoms of the targeted disease; (3) bringing about ameliorations of the symptoms of the targeted disease; (4) reducing the severity or incidence of the targeted disease; or (5) curing the targeted disease.
  • a therapeutically effective amount may be administered prior to the onset of the targeted disease, for a prophylactic or preventive action. Alternatively, or additionally, the therapeutically effective amount may be administered after initiation of the targeted disease, for a therapeutic action.
  • a therapeutically effective amount of the antibody (preferably the monoclonal antibody) of the present invention may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody (preferably the monoclonal antibody) of the present invention to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects.
  • the efficient dosages and dosage regimens for the antibody (preferably the monoclonal antibody) of the present invention depend on the disease or condition to be treated and may be determined by the persons skilled in the art. A physician having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • a suitable dose of a composition of the present invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect according to a particular dosage regimen.
  • Such an effective dose will generally depend upon the factors described above.
  • a therapeutically effective amount for therapeutic use may be measured by its ability to stabilize the progression of disease.
  • the ability of a compound to inhibit cancer may, for example, be evaluated in an animal model system predictive of efficacy in human tumors.
  • this property of a composition may be evaluated by examining the ability of the compound to induce cytotoxicity by in vitro assays known to the skilled practitioner.
  • a therapeutically effective amount of a therapeutic compound may decrease tumor size, or otherwise ameliorate symptoms in a subject.
  • One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected.
  • An exemplary, non-limiting range for a therapeutically effective amount of an antibody of the present invention is about 0.1-100 mg/kg, such as about 0.1-50 mg/kg, for example about 0.1-20 mg/kg, such as about 0.1-10 mg/kg, for instance about 0.5, about such as 0.3, about 1, about 3 mg/kg, about 5 mg/kg or about 8 mg/kg.
  • An exemplary, non-limiting range for a therapeutically effective amount of an antibody of the present invention is 0.02-100 mg/kg, such as about 0.02-30 mg/kg, such as about 0.05-10 mg/kg or 0.1-3 mg/kg, for example about 0.5-2 mg/kg.
  • Administration may e.g., be intravenous, intramuscular, intraperitoneal, or subcutaneous, and for instance administered proximal to the site of the target.
  • Dosage regimens in the above methods of treatment and uses are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.
  • the efficacy of the treatment is monitored during the therapy, e.g., at predefined points in time. In some embodiments, the efficacy may be monitored by visualization of the disease area, or by other diagnostic methods described further herein, e.g.
  • an effective daily dose of a pharmaceutical composition may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
  • the human antibody (preferably the monoclonal antibody) of the present invention are administered by slow continuous infusion over a long period, such as more than 24 hours, in order to minimize any unwanted side effects.
  • An effective dose of an antibody of the present invention may also be administered using a weekly, biweekly or triweekly dosing period.
  • the dosing period may be restricted to, e.g., 8 weeks, 12 weeks or until clinical progression has been established.
  • treatment according to the present invention may be provided as a daily dosage of an antibody of the present invention in an amount of about 0.1-100 mg/kg, such as 0.2, 0.5, 0.9, 1.0, 1.1 , 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, on at least one of days 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively, at least one of weeks 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 after initiation of treatment, or any combination
  • the antibody (preferably the monoclonal antibody) of the present invention is administered to the subject in the form of a pharmaceutical composition which comprises a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to an excipient that does not produce an adverse, allergic or other untoward reaction when administered to an animal, preferably a human. It includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
  • preparations should meet sterility, pyrogenicity, general safety and purity standards as required by regulatory offices, such as, for example, FDA Office or EMA.
  • compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene- block polymers, polyethylene glycol and wool fat.
  • ion exchangers alumina, aluminum stearate, lecithin
  • serum proteins such as human serum albumin
  • buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial g
  • compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir.
  • the used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.
  • Sterile injectable forms of the compositions of this invention may be aqueous or an oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1 ,3-butanediol.
  • a non-toxic parenterally acceptable diluent or solvent for example as a solution in 1 ,3-butanediol.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono-or diglycerides.
  • Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions.
  • compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers commonly used include lactose and corn starch.
  • Lubricating agents such as magnesium stearate, are also typically added.
  • useful diluents include, e.g., lactose.
  • the active ingredient is combined with emulsifying and suspending agents.
  • certain sweetening, flavoring or coloring agents may also be added.
  • the compositions of this invention may be administered in the form of suppositories for rectal administration. These can be prepared by mixing the agent with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release the drug.
  • suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release the drug.
  • Such materials include cocoa butter, beeswax and polyethylene glycols.
  • compositions of this invention may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs.
  • the compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers.
  • Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water.
  • compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers.
  • suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2- octyldodecanol, benzyl alcohol and water.
  • Topical application for the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. Patches may also be used.
  • the compositions of this invention may also be administered by nasal aerosol or inhalation.
  • compositions are prepared according to techniques well- known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents.
  • an antibody present in a pharmaceutical composition of this invention can be supplied at a concentration of 10 mg/mL in either 100 mg (10 mL) or 500 mg (50 mL) single-use vials.
  • the product is formulated for IV administration in 9.0 mg/mL sodium chloride, 7.35 mg/mL sodium citrate dihydrate, 0.7 mg/mL polysorbate 80, and Sterile Water for Injection. The pH is adjusted to 6.5.
  • An exemplary suitable dosage range for an antibody in a pharmaceutical composition of this invention may between about 1 mg/m 2 and 500 mg/m 2 .
  • schedules are exemplary and that an optimal schedule and regimen can be adapted taking into account the affinity and tolerability of the particular antibody in the pharmaceutical composition that must be determined in clinical trials.
  • a pharmaceutical composition of the invention for injection e.g. , intramuscular, i.v.
  • Another object of the invention is the use of at least one antibody of the present invention for detecting CD 160-TM in a sample, preferably in a biological sample, in vitro or in vivo.
  • Another object of the invention is the use of at least one antibody of the present invention for detecting activated NK cells in a sample, preferably in a biological sample, in vitro or in vivo.
  • assays in which the antibody of the invention may be used include, but are not limited to, ELISA, sandwich ELISA, RIA, FACS, tissue immunohistochemistry, Western- blot, and immunoprecipitation.
  • Another object of the invention is a method for detecting CD 160-TM in a sample, comprising contacting the sample with an antibody of the invention and detecting the antibody bound to CD 160-TM, thereby indicating the presence of CD 160-TM in the sample.
  • Another object of the invention is a method for detecting activated NK cells in a sample, comprising contacting the sample with an antibody of the invention and detecting the antibody bound to CD 160-TM, thereby indicating the presence of activated NK cells in the sample.
  • the sample is a biological sample.
  • biological samples include, but are not limited to, tissue lysates and extracts prepared from diseased tissues, bodily fluids, preferably blood, more preferably blood serum, plasma, synovial fluid, bronchoalveolar lavage fluid, sputum, lymph, ascitic fluids, urine, amniotic fluid, peritoneal fluid, cerebrospinal fluid, pleural fluid, pericardial fluid, and alveolar macrophages.
  • sample is intended to mean a sample taken from an individual prior to any analysis.
  • the antibody of the invention is directly labeled with a detectable label and may be detected directly.
  • the antibody of the invention is unlabeled (and is referred as the first/primary antibody) and a secondary antibody or other molecule that can bind the anti-CD 160-TM antibody is labeled.
  • a secondary antibody is chosen to be able to specifically bind the specific species and class of the primary antibody.
  • the presence of anti-CD 160-TM antibody / CD 160 complex in the sample can be detected and measured by detecting the presence of the labeled secondary antibody. For example, after washing away unbound secondary antibody from a well comprising the primary antibody/antigen complex or from a membrane (such as a nitrocellulose or nylon membrane) comprising the complex, the bound secondary antibody can be developed and detected based on chemiluminescence of the label for example.
  • Labels for the anti-CD 160-TM antibody or the secondary antibody include, but are not limited to, various enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic agents and radioactive materials.
  • enzymes include but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase or acetylcholinesterase
  • prosthetic group complexes include but are not limited to, streptavidin/biotin and avidin/biotin
  • examples of fluorescent materials include but are not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyne chloride or phycoerythrin
  • luminescent material include but are not limited to, luminal
  • examples of magnetic agents include gadolinium
  • suitable radioactive material include 1251, 1311, 35S or 3H.
  • Another object of the invention is an in vitro method for depleting a cell sample or a cell population from CD 160-TM expressing cells (e.g., from activated NK cells), comprising contacting the cell sample with an antibody of the present invention.
  • Another object of the invention is an in vitro method for isolating CD 160-TM expressing cells (e.g., from activated NK cells) from a cell sample or a cell population, comprising contacting the cell sample with an antibody of the present invention.
  • Another object of the invention is an in vitro method for activating NK cells, comprising contacting NK cells with an antibody of the present invention.
  • Another object of the invention is a kit comprising at least one anti-CD 160-TM antibody of the invention, preferably a monoclonal anti-CD 160-TM antibody.
  • kit any manufacture (e.g., a package or a container) comprising at least one reagent, preferably an antibody, for specifically detecting the expression of CD 160-TM.
  • kit may be promoted, distributed, or sold as a unit for performing the methods of the present invention. Furthermore, any or all of the kit reagents may be provided within containers that protect them from the external environment, such as in sealed containers.
  • kits may also contain a package insert describing the kit and methods for its use.
  • FIGURES
  • Figure 1 Binding specificity of A12 and B6 antibodies on CD160-GPI vs CD160TM expressing cells.
  • Mouse or human isotype control Igs were used as negative controls (black histograms). Bound antibodies were revealed with the appropriate PE-conjugated secondary reagents.
  • FIG. 2 Binding specificity of A12 on IL2-treated human PBMC.
  • PBMC were either left untreated (Day 0) or incubated with IL2.
  • Immuno-labelling were performed at the indicated time points with either an isotypic contral IgG (black histogram) or A12 antibody (grey histogram) plus PE-coupled goat anti-human IgG antibodies.
  • Lymphocytes subsets were further identified by addition of a mix of CD8-FITC, CD56-PC5, CD3-APC and CD4-PC7 mAbs. Shown are the labellings obtained on each gated lymphocyte population.
  • Figure 3 Assessment of A12 specificity by immunoprecipitation. Post-nuclear lysates were prepared from HEK-CD160TM cells and subjected to immunoprecipitation with either mouse (mu IgG) or human (hu Ig) isotypic control IgG, chimeric murine A12 (mu A 12) or fully human A12 (hu A12) antibody. Immuno-precipitated proteins were separated by SDS- 10% PAGE under non-reducing conditions, transferred on nitrocellulose and revealed by Western blot using an anti-Flag mAb. Arrows indicate CD 160TM -related signals.
  • Figure 5 shows the alignments of VH and VL sequences of A12 and B6 antibodies.
  • Figure 6 shows the conformation epitope recognized A12 and B6 antibodies composes of 2 peptides.
  • the peptides are indicated in BOLD and UNDERLINED.
  • the different domain of the CD160-TM isoform are also represented.
  • Figure 7 A12 induces NK cells degranulation and activation.
  • the NK92 cell line was pre -incubated with isotype control mulgG or a chimeric Fc murine version of the human A12 antibody (muA12) plus rabbit anti-mouse IgG antibodies. Effector cells were then incubated in the presence of the NK sensible target cells (K562 cell line) at the indicated E/T ratio.
  • the NK cell line NK92 cell degranulation and activation was monitored by detection of membrane associated CD 107a (A) and CD 137 (B), respectively. Given are the percentages of positive cells pre -treated with mulgG control (circles) or muA12 (squares).
  • CHO or HEK cells were transfected with a eukaryotic expression vector encompassing CD160-GPI or Flag-tagged CD160-TM cDNA, respectively. Stable transfectants were obtained by selection with the appropriate antibiotic and named thereafter CHO-CD160-GPI and HEK- CD160TM. Proper expression of CD 160 isoforms was assessed by flow cytometry using the anti-CD 160-GPI specific mAb CL1R2 or anti-Flag mAb plus PE-coupled goat anti-mouse IgG.
  • PBMC peripheral blood mononuclear cells
  • Fully human aglycosylated anti-CD 160TM antibodies were selected by phage display on HEK-CD160TM cells.
  • A12 was identified as the one giving the best recognition profile on HEK-CD160TM cells by flow cytometry.
  • CHO and HEK transfected cells were labelled with the anti-CD 160-GPI mAb CL1-R2 or BY55, the fully human A12 or B6 antibody or their corresponding isotypic control IgG. Bound antibodies were further revealed by addition of PE-coupled goat anti-mouse or anti- human IgG. Cell acquisition was performed on a CytoFlex cytometer and results were analysed with Flow Jo software.
  • CD 160 labelling was performed as above. Following washes and addition of normal mouse serum, cells were incubated with a mix of CD8-FITC, CD56-PC5, CD3-APC and CD4-PC7 mAbs. After cell acquisition, analyses were performed to distinguish the CD3 CD4 + and CD3 + CD8 + T lymphocytes, and the CD3 CD56 + NK cells within the lymphocytes population.
  • HEK-CD160TM cells that express a Flag-tagged version of CD160TM isoform, were lysed in 1% NP40 lysis buffer. Post-nuclear lysates were prepared and subjected to immuno- precipitation with the fully human A12 antibody or mouse chimeric A 12. Human and mouse IgG were used as negative controls, respectively. Immune complexes were further collected with protein G Sepharose beads. Following washes, non-reducing sample buffer (devoid of reducing agent) was added and samples were finally heat-denatured. Proteins were separated by SDS-10% PAGE, electrically transferred on a nitrocellulose membrane and subjected to immuno-blotting with and anti-Flag mAb plus HRP-coupled goat anti-mouse IgGs. Revelation was performed by enhanced chemiluminescence and images acquired with an ImageQuant LAS device.
  • the use of the murine chimeric A12 antibody led to the detection of proteins bands with an apparent molecular weight of 34-38, 56 and 100 kDa, that most likely correspond to the mono-, di-, and quadrimeric form of the receptor, respectively.
  • B6 antibody also results from the phage display selection as described in EXAMPLE 1.
  • B6 was identified as giving a very good recognition profile on HEK-CD160TM cells by flow cytometry.
  • Figure 5 shows the alignment of the VH and VL sequences of A12 and B6 antibodies and we can conclude that B6 is very similar to A12.
  • EXAMPLE 3 CHARACTERIZATION OF THE EPITOPE RECOGNIZED BY A12 AND B6
  • Epitope mapping of A12 and B6 was performed according to published protocols (Sloostra et al, Mol. Divers. (1996), Timmerman et al, J. Mol. Recognit. 20 5L283-299 (2007)). Briefly, the binding of antibody to each peptide was tested in a PEPSCAN-based ELISA. Surprisingly, we found that the epitope recognized by A12 an B6 antibodies is a conformational epitope composed of 2 peptides: SEQ ID NO: 4 and SEQ ID NO: 5. Despite the fact that the first peptide is commonly shared by CD160-GPI and CD160TM, the second peptide is specific to CD160TM explaining the specificity of both B6 and A12 antibodies for the CD160-TM isoform.
  • CD160-TM Engagement of CD160-TM with an antibody of the present invention (muA12 antibody) enhances the expression of CD 137 and the cell cytotoxicity (expression of CD 107a) against K652 cells (figure 7). Best results are obtained at low E/T ratio (1/1 and 2.5/1). Similar results were obtained with muB6 antibody (data not shown).

Abstract

La présente invention concerne des anticorps monoclonaux qui se lient à l'isoforme CD160-TM. Les inventeurs ont mis au point de nouveaux anticorps monoclonaux qui se lient à l'isoforme CD160-TM, mais ne se lient pas à l'isoforme ancré CD160 GPI, non pas à l'isoforme soluble CD160. En particulier, les anticorps de la présente invention sont appropriés pour amplifier l'activation des cellules NK et par conséquent, les cellules NK à fonctions cytotoxiques.
PCT/EP2017/077261 2016-10-25 2017-10-25 Anticorps monoclonaux se liant à l'isoforme transmembranaire cd160 WO2018077926A1 (fr)

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AU2017351764A AU2017351764A1 (en) 2016-10-25 2017-10-25 Monoclonal antibodies binding to the CD160 transmembrane isoform
US16/343,889 US11186635B2 (en) 2016-10-25 2017-10-25 Monoclonal antibodies binding to the CD160 transmembrane isoform
CA3080270A CA3080270A1 (fr) 2016-10-25 2017-10-25 Anticorps monoclonaux se liant a l'isoforme transmembranaire cd160
JP2019542804A JP2019535306A (ja) 2016-10-25 2017-10-25 Ｃｄ１６０膜貫通型アイソフォームと結合するモノクローナル抗体
BR112019008345A BR112019008345A8 (pt) 2016-10-25 2017-10-25 Anticorpos monoclonais ligados à isoforma transmembrana cd160
CN201780080063.4A CN110121508A (zh) 2016-10-25 2017-10-25 与cd160跨膜同种型结合的单克隆抗体
EP17797084.5A EP3532502A1 (fr) 2016-10-25 2017-10-25 Anticorps monoclonaux se liant à l'isoforme transmembranaire cd160
KR1020197014634A KR20190084264A (ko) 2016-10-25 2017-10-25 Cd160 막 횡단 동형체에 결합하는 단클론 항체

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WO2020127377A1 (fr) 2018-12-21 2020-06-25 Ose Immunotherapeutics Molécule bifonctionnelle anti-pd-1/il -7
WO2020165374A1 (fr) 2019-02-14 2020-08-20 Ose Immunotherapeutics Molécule bifonctionnelle comprenant il-15ra
WO2021122866A1 (fr) 2019-12-17 2021-06-24 Ose Immunotherapeutics Molécules bifonctionnelles comprenant un variant de l'il-7
WO2022195241A1 (fr) * 2021-03-17 2022-09-22 Ucl Business Ltd Domaine de liaison à cd160
WO2022214652A1 (fr) 2021-04-09 2022-10-13 Ose Immunotherapeutics Échafaudage pour molécules bifonctionnelles comprenant des domaines de liaison pd-1 ou cd28 et sirp
WO2022214653A1 (fr) 2021-04-09 2022-10-13 Ose Immunotherapeutics Nouvel échafaudage pour molécules bifonctionnelles présentant des propriétés améliorées
WO2023170207A1 (fr) * 2022-03-09 2023-09-14 Alderaan Biotechnology Anticorps d'isoforme transmembranaire anti-cd160
WO2024028386A1 (fr) 2022-08-02 2024-02-08 Ose Immunotherapeutics Molécule multifonctionnelle dirigée contre cd28

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